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Sample records for aeruginosa pao1 strain

  1. Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains.

    PubMed

    Klockgether, Jens; Munder, Antje; Neugebauer, Jens; Davenport, Colin F; Stanke, Frauke; Larbig, Karen D; Heeb, Stephan; Schöck, Ulrike; Pohl, Thomas M; Wiehlmann, Lutz; Tümmler, Burkhard

    2010-02-01

    Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections. PMID:20023018

  2. Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1

    PubMed Central

    Damron, F. Heath; Davis, Michael R.; Withers, T. Ryan; Ernst, Robert K.; Goldberg, Joanna B.; Yu, Guangli; Yu, Hongwei D.

    2011-01-01

    Summary Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are nonmucoid producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing nonmucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants. PMID:21631603

  3. The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting

    PubMed Central

    Sana, Thibault G.; Berni, Benjamin; Bleves, Sophie

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process. PMID:27376031

  4. Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA, a Gene That Is Disrupted in the PAO1 Strain

    PubMed Central

    Gutu, Alina D.; Rodgers, Nicole S.; Park, Jihye

    2015-01-01

    The arn locus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. In Pseudomonas aeruginosa, several two-component regulatory systems (TCSs) control the arn locus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of the P. aeruginosa PAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety of P. aeruginosa strains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such as P. aeruginosa PAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designated cprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. The cprA gene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutated cprA allele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion of cprA did not affect the arn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance. PMID:26100714

  5. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates.

    PubMed Central

    Kureishi, A; Diver, J M; Beckthold, B; Schollaardt, T; Bryan, L E

    1994-01-01

    The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtilis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA: Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis. Images PMID:7811002

  6. Genes related to chromate resistance by Pseudomonas aeruginosa PAO1.

    PubMed

    Rivera, Sonia L; Vargas, Eréndira; Ramírez-Díaz, Martha I; Campos-García, Jesús; Cervantes, Carlos

    2008-08-01

    Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms. PMID:18446454

  7. Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain

    PubMed Central

    2012-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. Results A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. Conclusions Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients. PMID:22264352

  8. Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

    PubMed Central

    Malhotra, Sonal; Silo-Suh, Laura A.; Mathee, Kalai; Ohman, Dennis E.

    2000-01-01

    Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase

  9. Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms

    PubMed Central

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed. PMID:24465697

  10. Fructooligosacharides reduce Pseudomonas aeruginosa PAO1 pathogenicity through distinct mechanisms.

    PubMed

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed. PMID:24465697

  11. Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance

    PubMed Central

    Periasamy, Saravanan; Nair, Harikrishnan A. S.; Lee, Kai W. K.; Ong, Jolene; Goh, Jie Q. J.; Kjelleberg, Staffan; Rice, Scott A.

    2015-01-01

    Pseudomonas aeruginosa PAO1 produces three polysaccharides, alginate, Psl, and Pel that play distinct roles in attachment and biofilm formation for monospecies biofilms. Considerably less is known about their role in the development of mixed species biofilm communities. This study has investigated the roles of alginate, Psl, and Pel during biofilm formation of P. aeruginosa in a defined and experimentally informative mixed species biofilm community, consisting of P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Loss of the Psl polysaccharide had the biggest impact on the integration of P. aeruginosa in the mixed species biofilms, where the percent composition of the psl mutant was significantly lower (0.06%) than its wild-type (WT) parent (2.44%). In contrast, loss of the Pel polysaccharide had no impact on mixed species biofilm development. Loss of alginate or its overproduction resulted in P. aeruginosa representing 8.4 and 18.11%, respectively, of the mixed species biofilm. Dual species biofilms of P. aeruginosa and K. pneumoniae were not affected by loss of alginate, Pel, or Psl, while the mucoid P. aeruginosa strain achieved a greater biomass than its parent strain. When P. aeruginosa was grown with P. protegens, loss of the Pel or alginate polysaccharides resulted in biofilms that were not significantly different from biofilms formed by the WT PAO1. In contrast, overproduction of alginate resulted in biofilms that were comprised of 35–40% of P. aeruginosa, which was significantly higher than the WT (5–20%). Loss of the Psl polysaccharide significantly reduced the percentage composition of P. aeruginosa in dual species biofilms with P. protegens (<1%). Loss of the Psl polysaccharide significantly disrupted the communal stress resistance of the three species biofilms. Thus, the polysaccharide composition of an individual species significantly impacts mixed species biofilm development and the emergent properties of such communities. PMID

  12. Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

    PubMed Central

    Ravichandran, Ayshwarya; Wong, Chui Ching; Swarup, Sanjay

    2015-01-01

    Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness. PMID:25894344

  13. In vitro antibiofilm activity of the freshwater bryozoan Hyalinella punctata: a case study of Pseudomonas aeruginosa PAO1.

    PubMed

    Pejin, Boris; Ciric, Ana; Karaman, Ivo; Horvatovic, Mladen; Glamoclija, Jasmina; Nikolic, Milos; Sokovic, Marina

    2016-08-01

    The antibiofilm and possible antiquorum sensing effects against the strain Pseudomonas aeruginosa PAO1 of five crude extracts of the freshwater bryozoan Hyalinella punctata (Hancock, 1850) were evaluated in vitro for the first time. H. punctata ethyl acetate extract (HpEtAc) exhibited the highest antibiofilm activity reducing the biofilm formation of P. aeruginosa PAO1 in the range of 80.63-88.13%. While all tested extracts reduced the twitching motility of the aforementioned bacterial strain, HpEtAc showed to be the most effective. Finally, at a concentration of 0.5 MIC, the same extract mostly inhibited the production of pyocyanin by P. aeruginosa PAO1 (71.53%). In comparison both with the positive controls used (streptomycin and ampicillin, 67.13 and 69.77%, respectively), HpEtAc was found to inhibit pyocyanin in a higher extent. An extensive chemical characterisation of this particular extract may result in isolation and identification of novel lead compounds targeting P. aeruginosa, an opportunistic human pathogen. PMID:26264659

  14. Pseudomonas aeruginosa PAO-1 Lipopolysaccharide-Diphtheria Toxoid Conjugate Vaccine: Preparation, Characterization and Immunogenicity

    PubMed Central

    Najafzadeh, Faezeh; Shapouri, Reza; Rahnema, Mehdi; Rokhsartalab Azar, Shadi; Kianmehr, Anvarsadat

    2015-01-01

    Background: Treatment of Pseudomonas aeruginosa PAO-1 infections through immunological means has been proved to be efficient and protective. Objectives: The purpose of this study was to produce a conjugate vaccine composed of detoxified lipopolysaccharide (D-LPS) P. aeruginosa and diphtheria toxoid (DT). Materials and Methods: Firstly, LPS was purified and characterized from P. aeruginosa PAO1 and then detoxified. D-LPS was covalently coupled to DT as a carrier protein via amidation method with adipic acid dihydrazide (ADH) as a spacer molecule and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) as a linker. The molar ratio of LPS to DT in the prepared conjugate was 3:1. The immunogenicity of D-LPS-DT conjugate vaccine in mice model was evaluated as well. Results: The conjugate was devoid of endotoxin activity and 0.125 U/mL of D-LPS was acceptable for immunization. D-LPS-DT conjugate was nonpyrogenic for rabbits and nontoxic for mice. Mice immunization with D-LPS-DT conjugate vaccine elicited the fourfold higher IgG antibody compared to D-LPS. Anti-LPS IgG antibody was predominantly IgG1 subclass and then IgG3, IgG2a and IgG2b, respectively. Conclusions: Vaccine based on the conjugation of P. aeruginosa PAO-1 LPS with DT increased anti-LPS antibodies and had a significant potential to protect against Pseudomonas infections. PMID:26301059

  15. Extracts of Cordia gilletii de wild (Boraginaceae) quench the quorum sensing of Pseudomonas aeruginosa PAO1

    PubMed Central

    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

  16. Determining Multiple Responses of Pseudomonas aeruginosa PAO1 to an Antimicrobial Agent, Free Nitrous Acid.

    PubMed

    Gao, Shu-Hong; Fan, Lu; Peng, Lai; Guo, Jianhua; Agulló-Barceló, Míriam; Yuan, Zhiguo; Bond, Philip L

    2016-05-17

    Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent on a range of micro-organisms, especially in wastewater-treatment systems. However, the antimicrobial mechanism of FNA is largely unknown. Here, we report that the antimicrobial effects of FNA are multitargeted. The response of a model denitrifier, Pseudomnas aeruginosa PAO1 (PAO1), common in wastewater treatment, was investigated in the absence and presence of inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. This was achieved through coupling gene expression analysis, by RNA sequencing, and with a suite of physiological analyses. Various transcripts exhibited significant changes in abundance in the presence of FNA. Respiration was likely inhibited because denitrification activity was severely depleted, and decreased transcript levels of most denitrification genes occurred. As a consequence, the tricarboxylic acid (TCA) cycle was inhibited due to the lowered cellular redox state in the FNA-exposed cultures. Meanwhile, during FNA exposure, PAO1 rerouted its carbon metabolic pathway from the TCA cycle to pyruvate fermentation with acetate as the end product as a possible survival mechanism. Additionally, protein synthesis was significantly decreased, and ribosome preservation was evident. These findings improve our understanding of PAO1 in response to FNA and contribute toward the potential application for use of FNA as an antimicrobial agent. PMID:27116299

  17. Transcriptome analysis of Pseudomonas aeruginosa PAO1 grown at both body and elevated temperatures.

    PubMed

    Chan, Kok-Gan; Priya, Kumutha; Chang, Chien-Yi; Abdul Rahman, Ahmad Yamin; Tee, Kok Keng; Yin, Wai-Fong

    2016-01-01

    Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 °C) and an arbitrarily-selected temperature of 46 °C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 °C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. PMID:27547539

  18. Transcriptome analysis of Pseudomonas aeruginosa PAO1 grown at both body and elevated temperatures

    PubMed Central

    Priya, Kumutha; Chang, Chien-Yi; Abdul Rahman, Ahmad Yamin; Tee, Kok Keng; Yin, Wai-Fong

    2016-01-01

    Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 °C) and an arbitrarily-selected temperature of 46 °C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 °C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. PMID:27547539

  19. Pseudomonas aeruginosa PAO1 virulence factors and poplar tree response in the rhizosphere

    PubMed Central

    Attila, Can; Ueda, Akihiro; Cirillo, Suat L. G.; Cirillo, Jeffrey D.; Chen, Wilfred; Wood, Thomas K.

    2008-01-01

    Summary Whole‐transcriptome analysis was used here for the first time in the rhizosphere to discern the genes involved in the pathogenic response of Pseudomonas aeruginosa PAO1 as well as to discern the response of the poplar tree. Differential gene expression shows that 185 genes of the bacterium and 753 genes of the poplar tree were induced in the rhizosphere. Using the P. aeruginosatranscriptome analysis, isogenic knockout mutants, and two novel plant assays (poplar and barley), seven novel PAO1 virulence genes were identified (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150 and PA4295). The uncharacterized putative haemolysin repressor, PA2463, upon inactivation, resulted in greater poplar virulence and elevated haemolysis while this mutant remained competitive in the rhizosphere. In addition, disruption of the haemolysin gene itself (PA2462) reduced the haemolytic activity of P. aeruginosa, caused less cytotoxicity and reduced barley virulence, as expected. Inactivating PA1385, a putative glycosyl transferase, reduced both poplar and barley virulence. Furthermore, disrupting PA2663, a putative membrane protein, reduced biofilm formation by 20‐fold. Inactivation of PA3476 (rhlI) increased virulence with barley as well as haemolytic activity and cytotoxicity, so quorum sensing is important in plant pathogenesis. Hence, this strategy is capable of elucidating virulence genes for an important pathogen. PMID:21261818

  20. Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lundgren, Benjamin R.; Thornton, William; Dornan, Mark H.; Villegas-Peñaranda, Luis Roberto; Boddy, Christopher N.

    2013-01-01

    Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria. PMID:23457254

  1. Nanomechanical Response of Pseudomonas aeruginosa PAO1 Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Dutcher, John

    2013-03-01

    We have used an atomic force microscopy (AFM)-based creep deformation technique to study changes to the viscoelastic properties of individual Gram-negative Pseudomonas aeruginosa PAO1 cells as a function of time of exposure to two cationic peptides: polymyxin B (PMB), a cyclic antimicrobial peptide, and the structurally-related compound, polymyxin B nonapeptide (PMBN). The measurements provide a direct measure of the mechanical integrity of the bacterial cell envelope, and the results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots, which can be ascribed to different components within the bacterial cell. Time-resolved creep deformation experiments reveal abrupt changes to the viscoelastic properties of P. aeruginosa bacterial cells after exposure to both PMB and PMBN, with quantitatively different changes for the two cationic peptides. These measurements provide new insights into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells.

  2. A theoretical and experimental proteome map of Pseudomonas aeruginosa PAO1

    PubMed Central

    Lecoutere, Elke; Verleyen, Peter; Haenen, Steven; Vandersteegen, Katrien; Noben, Jean-Paul; Robben, Johan; Schoofs, Liliane; Ceyssens, Pieter-Jan; Volckaert, Guido; Lavigne, Rob

    2012-01-01

    A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks. PMID:22950023

  3. Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress.

    PubMed

    Orlandi, Viviana T; Bolognese, Fabrizio; Chiodaroli, Luca; Tolker-Nielsen, Tim; Barbieri, Paola

    2015-12-01

    Pseudomonas aeruginosa is an opportunistic pathogen known to be resistant to different classes of antibiotics and disinfectants. P. aeruginosa also displays a certain degree of tolerance to photodynamic therapy (PDT), an alternative antimicrobial approach exploiting a photo-oxidative stress induced by exogenous photosensitizers and visible light. To evaluate whether P. aeruginosa pigments can contribute to its relative tolerance to PDT, we analysed the response to this treatment of isogenic transposon mutants of P. aeruginosa PAO1 with altered pigmentation. In general, in the presence of pigments a higher tolerance to PDT-induced photo-oxidative stress was observed. Hyperproduction of pyomelanin makes the cells much more tolerant to stress caused by either radicals or singlet oxygen generated by different photosensitizers upon photoactivation. Phenazines, pyocyanin and phenazine-1-carboxylic acid, produced in different amounts depending on the cultural conditions, are able to counteract both types of PDT-elicited reactive oxygen species. Hyperproduction of pyoverdine, caused by a mutation in a quorum-sensing gene, rendered P. aeruginosa more tolerant to a photosensitizer that generates mainly singlet oxygen, although in this case the observed tolerance to photo-oxidative stress cannot be exclusively attributed to the presence of the pigment. PMID:26419906

  4. In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1

    PubMed Central

    Sedighi, Mansour; Moghoofei, Mohsen; Kouhsari, Ebrahim; Pournajaf, Abazar; Emadi, Behzad; Tohidfar, Masoud; Gholami, Mehrdad

    2015-01-01

    Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions. PMID:26989748

  5. Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370.

    PubMed

    Zaitseva, Julia; Granik, Vladimir; Belik, Alexandr; Koksharova, Olga; Khmel, Inessa

    2009-06-01

    Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25-100microg/ml, nitrofurans 2-2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3-6-fold. For B. cenocepacia 370, the enhancement was 2-5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively. PMID:19460431

  6. Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

    PubMed

    Herbst, Florian-Alexander; Søndergaard, Mads T; Kjeldal, Henrik; Stensballe, Allan; Nielsen, Per H; Dueholm, Morten S

    2015-01-01

    The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections. PMID:25317949

  7. Evaluation of Pseudomonas aeruginosa (PAO1) adhesion to human alveolar epithelial cells A549 using SYTO 9 dye.

    PubMed

    Larrosa, Mar; Truchado, Pilar; Espín, Juan Carlos; Tomás-Barberán, Francisco A; Allende, Ana; García-Conesa, María Teresa

    2012-06-01

    Pseudomonas aeruginosa is an aerobic Gram-negative bacterium characterized by a natural resistance to several antibiotics. It is a major cause of nosocomial infections in patients with compromised host defence mechanisms mainly related to the respiratory tract. P. aeruginosa infection first step is the adhesion of the bacteria to the host cells and thus, the development of techniques that can easily assess adhesion of bacteria strains and of bacteria isolated from biological samples is fundamental. The aim of our work was to develop a fast and effective method to evaluate the adhesion of P. aeruginosa to bronchial epithelial cells. To meet our goal we optimized a staining protocol using the vital dye SYTO 9 and P. aeruginosa PAO1. We established the appropriate dying conditions as well as the stability of the stained bacteria. Adhesion was first measured using the traditional plate counting method and then, adhesion values were compared to those obtained using a fluorescence microplate reader and epifluorescence microscopy. Our results show that the use of SYTO 9 does not interfere with the bacteria viability, bacteria cell growth, and adhesion of P. aeruginosa to A549 epithelial cells. Both the fluorescence microplate reader and the epifluorescence microscopy gave similar results to those attained with the plate counting method, however, the epifluorescence microscopy also allowed for simultaneous discrimination of damaging effects on the human cells. Overall, our data indicate that the use of SYTO 9 combined with a fluorescence microplate reader or an epifluorescence microscope provides a rapid method to evaluate the adhesion of P. aeruginosa to human epithelial cells. However, to show unequivocally that a specific drug or compound has a truly inhibitory effect on the bacterial adhesion without affecting the number of human cells, the epifluorescence microscopy is recommended. PMID:22464926

  8. Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1

    PubMed Central

    Betts, Alex; Vasse, Marie; Kaltz, Oliver; Hochberg, Michael E

    2013-01-01

    Antibiotic resistance is becoming increasingly problematic for the treatment of infectious disease in both humans and livestock. The bacterium Pseudomonas aeruginosa is often found to be resistant to multiple antibiotics and causes high patient mortality in hospitals. Bacteriophages represent a potential option to combat pathogenic bacteria through their application in phage therapy. Here, we capitalize on previous studies showing how evolution may increase phage infection capacity relative to ancestral genotypes. We passaged four different phage isolates (podoviridae, myoviridae) through six serial transfers on the ancestral strain of Pseudomonas aeruginosa PAO1. We first demonstrate that repeated serial passage on ancestral bacteria increases infection capacity of bacteriophage on ancestral hosts and on those evolved for one transfer. This result is confirmed when examining the ability of evolved phage to reduce ancestral host population sizes. Second, through interaction with a single bacteriophage for 24 h, P. aeruginosa can evolve resistance to the ancestor of that bacteriophage; this also provides these evolved bacteria with cross-resistance to the other three bacteriophages. We discuss how the evolutionary training of phages could be employed as effective means of combatting bacterial infections or disinfecting surfaces in hospital settings, with reduced risk of bacterial resistance compared with conventional methods. PMID:24187587

  9. Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1.

    PubMed

    Betts, Alex; Vasse, Marie; Kaltz, Oliver; Hochberg, Michael E

    2013-11-01

    Antibiotic resistance is becoming increasingly problematic for the treatment of infectious disease in both humans and livestock. The bacterium Pseudomonas aeruginosa is often found to be resistant to multiple antibiotics and causes high patient mortality in hospitals. Bacteriophages represent a potential option to combat pathogenic bacteria through their application in phage therapy. Here, we capitalize on previous studies showing how evolution may increase phage infection capacity relative to ancestral genotypes. We passaged four different phage isolates (podoviridae, myoviridae) through six serial transfers on the ancestral strain of Pseudomonas aeruginosa PAO1. We first demonstrate that repeated serial passage on ancestral bacteria increases infection capacity of bacteriophage on ancestral hosts and on those evolved for one transfer. This result is confirmed when examining the ability of evolved phage to reduce ancestral host population sizes. Second, through interaction with a single bacteriophage for 24 h, P. aeruginosa can evolve resistance to the ancestor of that bacteriophage; this also provides these evolved bacteria with cross-resistance to the other three bacteriophages. We discuss how the evolutionary training of phages could be employed as effective means of combatting bacterial infections or disinfecting surfaces in hospital settings, with reduced risk of bacterial resistance compared with conventional methods. PMID:24187587

  10. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1.

    PubMed

    Nithya, Chari; Begum, Mansur Farzana; Pandian, Shunmugiah Karutha

    2010-09-01

    According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 microg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70-74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat. PMID:20665017

  11. Kinetic modeling of rhamnolipid production by Pseudomonas aeruginosa PAO1 including cell density-dependent regulation.

    PubMed

    Henkel, Marius; Schmidberger, Anke; Vogelbacher, Markus; Kühnert, Christian; Beuker, Janina; Bernard, Thomas; Schwartz, Thomas; Syldatk, Christoph; Hausmann, Rudolf

    2014-08-01

    The production of rhamnolipid biosurfactants by Pseudomonas aeruginosa is under complex control of a quorum sensing-dependent regulatory network. Due to a lack of understanding of the kinetics applicable to the process and relevant interrelations of variables, current processes for rhamnolipid production are based on heuristic approaches. To systematically establish a knowledge-based process for rhamnolipid production, a deeper understanding of the time-course and coupling of process variables is required. By combining reaction kinetics, stoichiometry, and experimental data, a process model for rhamnolipid production with P. aeruginosa PAO1 on sunflower oil was developed as a system of coupled ordinary differential equations (ODEs). In addition, cell density-based quorum sensing dynamics were included in the model. The model comprises a total of 36 parameters, 14 of which are yield coefficients and 7 of which are substrate affinity and inhibition constants. Of all 36 parameters, 30 were derived from dedicated experimental results, literature, and databases and 6 of them were used as fitting parameters. The model is able to describe data on biomass growth, substrates, and products obtained from a reference batch process and other validation scenarios. The model presented describes the time-course and interrelation of biomass, relevant substrates, and products on a process level while including a kinetic representation of cell density-dependent regulatory mechanisms. PMID:24770383

  12. Identification of Novel Genes Responsible for Overexpression of ampC in Pseudomonas aeruginosa PAO1

    PubMed Central

    Tsutsumi, Yuko; Tomita, Haruyoshi

    2013-01-01

    The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to β-lactams. The expression of genes reported to be involved in β-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR. PMID:24041903

  13. The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1.

    PubMed

    Lin, Jinshui; Cheng, Juanli; Chen, Keqi; Guo, Chenghao; Zhang, Weipeng; Yang, Xu; Ding, Wei; Ma, Li; Wang, Yao; Shen, Xihui

    2015-01-01

    The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1. PMID:26484316

  14. The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lin, Jinshui; Cheng, Juanli; Chen, Keqi; Guo, Chenghao; Zhang, Weipeng; Yang, Xu; Ding, Wei; Ma, Li; Wang, Yao; Shen, Xihui

    2015-01-01

    The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1. PMID:26484316

  15. Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1

    PubMed Central

    Li, Shanshan; Li, Tingting; Xu, Yueyang; Zhang, Qionglin; Zhang, Wei; Che, Shiyou; Liu, Ruihua; Wang, Yingying; Bartlam, Mark

    2015-01-01

    YfiBNR is a tripartite signalling system in Pseudomonas aeruginosa that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiB is an outer membrane lipoprotein and presumed sensor protein that sequesters the repressor protein YfiR. To provide insights into YfiBNR function, we have determined three-dimensional crystal structures of YfiB and YfiR from P. aeruginosa PAO1 alone and as a 1:1 complex. A YfiB(27–168) construct is predominantly dimeric, whereas a YfiB(59–168) is monomeric, indicating that YfiB can dimerize via its N-terminal region. YfiR forms a stable complex with YfiB(59–168), while the YfiR binding interface is obstructed by the N-terminal region in YfiB(27–168). The YfiB-YfiR complex reveals a conserved interaction surface on YfiR that overlaps with residues predicted to interact with the periplasmic PAS domain of YfiN. Comparison of native and YfiR-bound structures of YfiB suggests unwinding of the N-terminal linker region for attachment to the outer membrane. A model is thus proposed for YfiR sequestration at the outer membrane by YfiB. Our work provides the first detailed insights into the interaction between YfiB and YfiR at the molecular level and is a valuable starting point for further functional and mechanistic studies of the YfiBNR signalling system. PMID:26593397

  16. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution. PMID:26957519

  17. FUNCTIONAL ANALYSIS OF GENES FOR BIOSYNTHESIS OF PYOCYANIN AND PHENAZINE-1-CARBOXAMIDE FROM PSEUDOMONAS AERUGINOSA PAO1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from P. fluorescens and P. aureofaciens. Functional studies in phenazine-nonpr...

  18. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You

    2016-01-01

    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa. PMID:27175902

  19. Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

    PubMed Central

    Lotlikar, Shalaka R.; Hnatusko, Shane; Dickenson, Nicholas E.; Choudhari, Shyamal P.; Picking, Wendy L.

    2013-01-01

    Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28–45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn2+ and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19–26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism. PMID:23728627

  20. Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1.

    PubMed

    Yamano, Y; Nishikawa, T; Komatsu, Y

    1993-05-01

    The porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N-terminal 23-amino-acid sequence and the reaction with anti-OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated E sigma 54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids. PMID:8394980

  1. Inhibition of Pseudomonas aeruginosa PAO1 adhesion to and invasion of A549 lung epithelial cells by natural extracts.

    PubMed

    Ahmed, Ghada F; Elkhatib, Walid F; Noreddin, Ayman M

    2014-01-01

    Pseudomonas aeruginosa colonizes the lungs in cystic fibrosis (CF) and mechanically ventilated patients by binding to the cellular receptors on the surface of the lung epithelium. Studies have shown that blocking this interaction could be achieved with sub-minimum inhibitory concentrations of antibiotics such as ciprofloxacin. The development of bacterial resistance is a probable drawback of such an intervention. The use of natural extracts to interfere with bacterial adhesion and invasion has recently gained substantial attention and is hypothesized to inhibit bacterial binding and consequently prevent or reduce pathogenicity. This study used an A549 lung epithelial cell infection model, and the results revealed that a combination of aqueous cranberry extract with ciprofloxacin could completely prevent the adhesion and invasion of P. aeruginosa PAO1 compared to the untreated control. All of the natural extracts (cranberry, dextran, and soybean extracts) and ciprofloxacin showed a significant reduction (P<0.0001) in P. aeruginosa PAO1 adhesion to and invasion of lung epithelial cells relative to the control. The cranberry, dextran, and soybean extracts could substantially increase the anti-adhesion and anti-invasion effects of ciprofloxacin to the averages of 100% (P<0.0001), 80% (P<0.0001), and 60% (P<0.0001), respectively. Those extracts might result in a lower rate of the development of bacterial resistance; they are relatively safe and inexpensive agents, and utilizing such extracts, alone or in combination with ciprofloxacin, as potential anti-adhesion and anti-invasion remedies, could be valuable in preventing or reducing P. aeruginosa lung infections. PMID:24894307

  2. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase.

    PubMed

    Van Alst, Nadine E; Sherrill, Lani A; Iglewski, Barbara H; Haidaris, Constantine G

    2009-10-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  3. A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry.

    PubMed

    Park, Amber J; Murphy, Kathleen; Krieger, Jonathan R; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M

    2014-04-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  4. A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry*

    PubMed Central

    Park, Amber J.; Murphy, Kathleen; Krieger, Jonathan R.; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M.

    2014-01-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  5. Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines

    PubMed Central

    Vázquez-Rivera, Dolores; González, Omar; Guzmán-Rodríguez, Jaquelina; Díaz-Pérez, Alma L.; Ochoa-Zarzosa, Alejandra; López-Bucio, José; Meza-Carmen, Víctor; Campos-García, Jesús

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10−5 and 1.8 × 10−4 mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix. PMID:25821788

  6. Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d-Amino Acid Mixture

    PubMed Central

    Sanchez, Zoe; Tani, Akio

    2013-01-01

    Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a d-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells. PMID:23220960

  7. Dictyostelium discoideum as a surrogate host-microbe model for antivirulence screening in Pseudomonas aeruginosa PAO1.

    PubMed

    Bravo-Toncio, Catalina; Álvarez, Javiera A; Campos, Francisca; Ortíz-Severín, Javiera; Varas, Macarena; Cabrera, Ricardo; Lagos, Carlos F; Chávez, Francisco P

    2016-05-01

    The interest of the pharmaceutical industry in developing new antibiotics is decreasing, as established screening systems which identify compounds that kill or inhibit the growth of bacteria can no longer be used. Consequently, antimicrobial screening using classical minimum inhibitory concentration (MIC) measurements is becoming obsolete. The discovery of antimicrobial agents that specifically target a bacterial pathogen without affecting the host and its beneficial bacteria is a promising strategy. However, few host-microbe models are available for in vivo screening of novel antivirulence molecules. Here we designed high-throughput developmental assays in the social amoeba Dictyostelium discoideum to measure Pseudomonas aeruginosa virulence and to screen for novel antivirulence molecules without side effects to the host and its beneficial bacteria Klebsiella aerogenes. Thirty compounds were evaluated that had been previously selected by virtual screening for inhibitors of P. aeruginosa PAO1 polyphosphate kinase 1 (PaPPK1) and diverse compounds with combined PPK1 inhibitory and antivirulence activities were identified. This approach demonstrates that D. discoideum is a suitable surrogate host for preliminary high-throughput screening of antivirulence agents and that PPK1 is a suitable target for developing novel antivirulence compounds that can be further validated in mammalian models. PMID:27066943

  8. Structural Analysis of WbpE from Pseudomonas aeruginosa PAO1: A Nucleotide Sugar Aminotransferase Involved in O-Antigen Assembly

    SciTech Connect

    Larkin, A.; Olivier, N; Imperiali, B

    2010-01-01

    In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5{prime}-phosphate (PLP) and product UDP-GlcNAc(3NH{sub 2})A as the external aldimine at 1.9 {angstrom} resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the {alpha}-carboxylate moiety of {alpha}-ketoglutarate, is also uniquely positioned to bestow specificity for the 6{double_prime}-carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6{double_prime}-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6{double_prime} atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2{double_prime}, C3{double_prime}, and C6{double_prime} positions and provides insight into a novel target for treatment of P

  9. Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1.

    PubMed

    Winder, C L; Al-Adham, I S; Abdel Malek, S M; Buultjens, T E; Horrocks, A J; Collier, P J

    2000-08-01

    Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT). PMID:10971761

  10. Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1

    PubMed Central

    Zincke, Diansy; Balasubramanian, Deepak; Silver, Lynn L.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter β-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function. PMID:26621621

  11. Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1.

    PubMed

    Rajesh, P S; Ravishankar Rai, V

    2014-01-01

    Quorum sensing mechanism allows the microorganisms to resist the antibiotic treatment by forming biofilms. Quorum quenching is one of the mechanisms to control the development of drug resistance in microbes. Endophyte bacteria are beneficial to plant growth as they support the immune system against the pathogen attack. The endophytic bacteria present in Pterocarpus santalinus were screened for the presence of N-acyl homoserine lactones (AHLs) degrading bacteria using biosensor strains and further confirmed by quantifying the violacein production. Cell-free lysate of endophytic bacteria, Bacillus firmus PT18 and Enterobacter asburiae PT39 exhibited potent AHL degrading ability by inhibiting about 80% violacein production in biosensor strain. Furthermore, when the cell-free lysate was applied to Pseudomonas aeruginosa PAO1 and PAO1-JP2 biofilm it resulted in significant (p<0.01) inhibition of biofilm formation. The biofilm inhibition was confirmed by visualization of biofilm slides under fluorescence microscopy, which showed decrease in total biomass formation in treated slides. Isolation and amplification of the gene (aiiA) indicated that the presence of AHL lactonase in cell-free lysate and sequence alignment indicated that AiiA contains a "HXHXDH" zinc-binding motif that is being conserved in several groups of metallohydrolases. Therefore, the study shows the potential of AHLs degradation by AHL lactonase present in cell-free lysate of isolated endophytic bacteria and inhibition of quorum sensing regulated biofilm formation in P. aeruginosa PAO1. PMID:24268182

  12. Growth-dependent alterations in production of serotype-specific and common antigen lipopolysaccharides in Pseudomonas aeruginosa PAO1.

    PubMed Central

    McGroarty, E J; Rivera, M

    1990-01-01

    Pseudomonas aeruginosa PAO1 was grown in various media and at different temperatures, and the heterogeneity of the extracted lipopolysaccharide (LPS) was characterized by polyacrylamide gel electrophoresis. The size distributions of the serotype-specific LPS and the common antigen LPS were analyzed on Western blots (immunoblots). Cells grown at high, near-growth-limiting temperatures, at low pH, in low concentrations of phosphate, or in high concentrations of NaCl, MgCl2, glycerol, or sucrose produced decreased amounts of the very long chain population of O-antigen LPS molecules. Lower temperatures and lowered glycerol, lowered sucrose, low sulfate, lower salt concentrations, and elevated pH did not significantly affect the level of this LPS population. The size and amount of common antigen LPS was either unaffected or increased slightly when the cells were grown under the above stress conditions. Cells grown under normal, nonstressed conditions were agglutinated only by serotype-specific antibodies. In contrast, cells grown under stress conditions, in which the long-O-polymer LPS was absent, were agglutinated by both serotype-specific and common antigen-specific antibodies. The results indicate that the long O polymers cover and mask the shorter common antigen. However, specific growth conditions limit the production of the long O polymer, allowing the exposure and reactivity of the common antigen on the cell surface. Images PMID:2108085

  13. Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

    PubMed Central

    Yang, Zhe; Lu, Chung-Dar

    2007-01-01

    The arginine transaminase (ATA) pathway represents one of the multiple pathways for l-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD+ and l-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated Vmax and kcat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s−1. The apparent Km and catalytic efficiency (kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM−1 s−1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM−1 s−1 for l-arginine. When l-lysine was used as the substrate, MS analysis suggested Δ1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism. PMID:17416668

  14. Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1.

    PubMed

    Schmidberger, Anke; Henkel, Marius; Hausmann, Rudolf; Schwartz, Thomas

    2014-08-01

    Bioprocesses based on sustainable resources and rhamnolipids in particular have become increasingly attractive in recent years. These surface-active glycolipids with various chemical and biological properties have diverse biotechnological applications and are naturally produced by Pseudomonas aeruginosa. Their production, however, is tightly governed by a complex growth-dependent regulatory network, one of the major obstacles in the way to upscale production. P. aeruginosa PAO1 was grown in shake flask cultures using varying concentrations of ferric iron. Gene expression was assessed using quantitative PCR. A strong increase in relative expression of the genes for rhamnolipid synthesis, rhlA and rhlC, as well as the genes of the pqs quorum sensing regulon was observed under iron-limiting conditions. Iron repletion on the other hand caused a down-regulation of those genes. Furthermore, gene expression of different iron regulation-related factors, i.e. pvdS, fur and bqsS, was increased in response to iron limitation. Ensuing from these results, a batch cultivation using production medium without any addition of iron was conducted. Both biomass formation and specific growth rates were not impaired compared to normal cultivation conditions. Expression of rhlA, rhlC and pvdS, as well as the gene for the 3-oxo-C12-HSL synthetase, lasI, increased until late stationary growth phase. After this time point, their expression steadily decreased. Expression of the C4-HSL synthetase gene, rhlI, on the other hand, was found to be highly increased during the entire process. PMID:24752844

  15. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production. PMID:26922727

  16. Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1.

    PubMed

    Morita, Yuji; Murata, Takeshi; Mima, Takehiko; Shiota, Sumiko; Kuroda, Teruo; Mizushima, Tohru; Gotoh, Naomasa; Nishino, Takeshi; Tsuchiya, Tomofusa

    2003-04-01

    Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently. PMID:12654738

  17. Molecular Characterization and Regulation of the aguBA Operon, Responsible for Agmatine Utilization in Pseudomonas aeruginosa PAO1

    PubMed Central

    Nakada, Yuji; Jiang, Ying; Nishijyo, Takayuki; Itoh, Yoshifumi; Lu, Chung-Dar

    2001-01-01

    Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (Mr 32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (Mr 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (Mr 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. PMID:11673419

  18. Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1.

    PubMed

    Nakada, Y; Jiang, Y; Nishijyo, T; Itoh, Y; Lu, C D

    2001-11-01

    Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. PMID:11673419

  19. Function-Biased Choice of Additives for Optimization of Protein Crystallization: The Case of the Putative Thioesterase PA5185 from Pseudomonas aeruginosa PAO1

    SciTech Connect

    Chruszcz, Maksymilian; Zimmerman, Matthew D.; Wang, Shuren; Koclega, Katarzyna D.; Zheng, Heping; Evdokimova, Elena; Kudritska, Marina; Cymborowski, Marcin; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2009-09-15

    The crystal structure of PA5185, a putative thioesterase from Pseudomonas aeruginosa strain PAO1, was solved using multi-wavelength anomalous diffraction to 2.4 {angstrom}. Analysis of the structure and information about the putative function of the protein were used to optimize crystallization conditions. The crystal growth was optimized by applying additives with chemical similarity to a fragment of a putative PA5185 substrate (CoA or its derivative). Using new crystallization conditions containing this function-biased set of additives, several new crystal forms were produced, and structures of three of them (in three different space groups) were determined. One of the new crystal forms had an improved resolution limit of 1.9 {angstrom}, and another displayed an alternative conformation of the highly conserved loop containing Asn26, which could play a physiological role. Surprisingly, none of the additives were ordered in the crystal structures. Application of function-biased additives could be used as a standard optimization protocol for producing improved diffraction, or new crystal forms, which may lead to better understanding of the biological functions of proteins.

  20. Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

    PubMed Central

    Sarabhai, Sajal; Sharma, Prince; Capalash, Neena

    2013-01-01

    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-C12HSL and C4HSL 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 C4HSL. F7 also showed antagonistic activity against 3-oxo-C12HSL-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 and enhanced

  1. Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1.

    PubMed

    Kim, Eun-Jin; Sabra, Wael; Zeng, An-Ping

    2003-09-01

    Pseudomonas aeruginosa PAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface. In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena. The physiological responses of two P. aeruginosa PAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations. In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed. The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient, kLa, significantly decreased under iron-limited conditions. The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration. The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way for P. aeruginosa PAO1 to adapt to growth conditions of iron limitation. Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation. The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions. These results have implications for lung infection of cystic fibrosis patients by P. aeruginosa in view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death. PMID:12949186

  2. Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1

    PubMed Central

    Yang, Wen; Li, Kan; Bai, Yuwei; Xu, Yueyang; Jin, Jin; Wang, Yingying; Bartlam, Mark

    2015-01-01

    Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation. PMID:25909591

  3. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1.

    PubMed

    Cui, Qinna; Lv, Huinan; Qi, Zhuangzhuang; Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  4. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  5. Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis.

    PubMed

    Choi, Ji Young; Sifri, Costi D; Goumnerov, Boyan C; Rahme, Laurence G; Ausubel, Frederick M; Calderwood, Stephen B

    2002-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA. PMID:11807055

  6. GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1

    PubMed Central

    Sarwar, Zaara; Lundgren, Benjamin R.; Grassa, Michael T.; Wang, Michael X.; Gribble, Megan; Moffat, Jennifer F.

    2016-01-01

    ABSTRACT Glycine serves as a major source of single carbon units for biochemical reactions within bacterial cells. Utilization of glycine is tightly regulated and revolves around a key group of proteins known as the glycine cleavage system (GCS). Our lab previously identified the transcriptional regulator GcsR (PA2449) as being required for catabolism of glycine in the opportunistic pathogen Pseudomonas aeruginosa PAO1. In an effort to clarify and have an overall better understanding of the role of GcsR in glycine metabolism, a combination of transcriptome sequencing and electrophoretic mobility shift assays was used to identify target genes of this transcriptional regulator. It was found that GcsR binds to an 18-bp consensus sequence (TGTAACG-N4-CGTTCCG) upstream of the gcs2 operon, consisting of the gcvH2, gcvP2, glyA2, sdaA, and gcvT2 genes. The proteins encoded by these genes, namely, the GCS (GcvH2-GcvP2-GcvT2), serine hydroxymethyltransferase (GlyA2), and serine dehydratase (SdaA), form a metabolic pathway for the conversion of glycine into pyruvate, which can enter the central metabolism. GcsR activates transcription of the gcs2 operon in response to glycine. Interestingly, GcsR belongs to a family of transcriptional regulators known as TyrR-like enhancer-binding proteins (EBPs). Until this study, TyrR-like EBPs were only known to function in regulating aromatic amino acid metabolism. GcsR is the founding member of a new class of TyrR-like EBPs that function in the regulation of glycine metabolism. Indeed, homologs of GcsR and its target genes are present in almost all sequenced genomes of the Pseudomonadales order, suggesting that this genetic regulatory mechanism is a common theme for pseudomonads. IMPORTANCE Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux

  7. Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1

    NASA Astrophysics Data System (ADS)

    Islam, Salim T.; Huszczynski, Steven M.; Nugent, Timothy; Gold, Alexander C.; Lam, Joseph S.

    2013-12-01

    O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein. Amino acid substitutions in CL6 were found to alter Wzz-mediated O-antigen modality, with evidence suggesting that these changes may perturb the C-terminal WzyPa tertiary structure. Together, these data suggest that the catch-and-release mechanism of O-Ag polymerization is widespread among bacteria and that regulation of polymer length is affected by interaction of Wzz with Wzy.

  8. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli.

    PubMed

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-08-31

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser(78) to Cys(78) resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys(78) in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  9. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

    PubMed Central

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-01-01

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser78 to Cys78 resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys78 in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  10. Regulation and characterization of the dadRAX locus for D-amino acid catabolism in Pseudomonas aeruginosa PAO1.

    PubMed

    He, Weiqing; Li, Congran; Lu, Chung-Dar

    2011-05-01

    D-amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the D-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for D- and L-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all D-amino acids tested as substrates except D-Glu and D-Gln. DadA also showed comparable k(cat) and K(m) values on D-Ala and several D-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of L-Ala, L-Trp, D-Ala, and a specific set of D-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on D-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several L-amino acids, most strongly by Ala, and only by D-Ala among all tested D-amino acids. Enhanced growth of the dadX mutant on D-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous D-Ala but not L-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of L-Ala but not D-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed D-amino acid dehydrogenase activity of broad substrate

  11. The Combined Structural and Kinetic Characterization of a Bacterial Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes NMO Class I and II*

    PubMed Central

    Salvi, Francesca; Agniswamy, Johnson; Yuan, Hongling; Vercammen, Ken; Pelicaen, Rudy; Cornelis, Pierre; Spain, Jim C.; Weber, Irene T.; Gadda, Giovanni

    2014-01-01

    Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBankTM as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with kcatapp/Kmapp of 12 × 106 m−1 s−1, kcatapp of 1300 s−1, and Kmapp of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO. PMID:25002579

  12. Functional analysis and regulation of the divergent spuABCDEFGH-spuI operons for polyamine uptake and utilization in Pseudomonas aeruginosa PAO1.

    PubMed

    Lu, Chung-Dar; Itoh, Yoshifumi; Nakada, Yuji; Jiang, Ying

    2002-07-01

    A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1. This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization). The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants. Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function. Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator. PMID:12081945

  13. Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-antigen Assembly in Pseudomonas aeruginosa PAO1

    PubMed Central

    Larkin, Angelyn; Imperiali, Barbara

    2009-01-01

    The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-d-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH2)A in a coupled reaction via a unique NAD+ recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH2)A to give UDP-GlcNAc(3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of LPS assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains. PMID:19348502

  14. Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

    PubMed Central

    Sharma, Vandana; Noriega, Chris E.; Rowe, John J.

    2006-01-01

    Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ΔnarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ΔnarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions. PMID:16391109

  15. Biological cost of hypermutation in Pseudomonas aeruginosa strains from patients with cystic fibrosis.

    PubMed

    Montanari, Sara; Oliver, Antonio; Salerno, Paola; Mena, Ana; Bertoni, Giovanni; Tümmler, Burkhard; Cariani, Lisa; Conese, Massimo; Döring, Gerd; Bragonzi, Alessandra

    2007-05-01

    The high prevalence of hypermutable (mismatch repair-deficient) Pseudomonas aeruginosa strains in patients with cystic fibrosis (CF) is thought to be driven by their co-selection with adaptive mutations required for long-term persistence. Whether the increased mutation rate of naturally hypermutable strains is associated with a biological benefit or cost for the colonization of secondary environments is not known. Thirty-nine P. aeruginosa strains were collected from ten patients with CF during their course of chronic lung infections and screened for hypermutability. Seven hypermutable P. aeruginosa strains (18 %) isolated from six patients with CF (60 %) were identified and assigned to five different genotypes. Complementation and sequence analysis in the mutS, mutL and uvrD genes of these hypermutable P. aeruginosa strains revealed novel mutations. To understand the consequences of hypermutation for the fitness of the organisms, five pairs of clinical wild-type/hypermutable, clonally related P. aeruginosa strains and the laboratory strains PAO1/PAO1DeltamutS were subjected to competition in vitro and in the agar-beads mouse model of chronic airway infection. When tested in competition assay in vitro, the wild-type outcompeted four clinical hypermutable strains and the PAO1DeltamutS strain. In vivo, all of the hypermutable strains were less efficient at establishing lung infection than their wild-type clones. These results suggest that P. aeruginosa hypermutation is associated with a biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility. PMID:17464058

  16. Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1.

    PubMed

    Choi, Sung-Chan; Zhang, Can; Moon, Sooyoung; Oh, Young-Sook

    2014-09-01

    4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development. PMID:25085732

  17. Identification of greA encoding a transcriptional elongation factor as a member of the carA-orf-carB-greA operon in Pseudomonas aeruginosa PAO1.

    PubMed Central

    Lu, C D; Kwon, D H; Abdelal, A T

    1997-01-01

    A homolog of the transcriptional elongation factor, GreA, was identified in Pseudomonas aeruginosa PAO1. The deduced amino acid sequence for GreA from this organism exhibits 65.2% identity to its counterpart in Escherichia coli K-12. The nucleotide sequence of greA from P. aeruginosa overlaps by four bases the 3' terminus of carB which encodes the large subunit of carbamoylphosphate synthetase. S1 nuclease experiments showed that level of the greA transcript is elevated approximately 10-fold under conditions of pyrimidine limitation, consistent with the conclusion that transcription is initiated from the previously identified pyrimidine-sensitive promoter upstream of the carA-orf-carB-greA operon. Transcriptional fusion experiments showed the presence of an additional weak promoter within the carB sequence. A greA insertional mutant of Pseudomonas aerugionsa was constructed by gene replacement. The mutant derivative grew well in rich medium but did not grow in minimal medium supplemented by arginine and nucleosides. The greA phenotype was suppressed by secondary mutations at a relatively high rate, consistent with the notion of an important physiological role for GreA. PMID:9139926

  18. Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-d-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-d-Rhamnosyltransferase WbpZ

    PubMed Central

    Wang, Shuo; Hao, Youai; Lam, Joseph S.; Vlahakis, Jason Z.; Szarek, Walter A.; Vinnikova, Anna; Veselovsky, Vladimir V.

    2015-01-01

    ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of d-rhamnose (d-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-d-Rha and enzymatically synthesized GDP-d-[3H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one d-Rha residue from GDP-d-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring d-mannose (d-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-d-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase that has significant activity of GDP-d-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a d-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. IMPORTANCE This is the first characterization of a d-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. PMID:25845842

  19. Insights into Mechanisms and Proteomic Characterisation of Pseudomonas aeruginosa Adaptation to a Novel Antimicrobial Substance

    PubMed Central

    Cierniak, Peter; Jübner, Martin; Müller, Stefan; Bender, Katja

    2013-01-01

    Antibiotic resistance has been reported since the introduction of synthetic antibiotics. Bacteria, such as one of the most common nosocomial pathogens P. aeruginosa, adapt quickly to changing environmental conditions, due to their short generation time. Thus microevolutional changes can be monitored in situ. In this study, the microevolutional process of Pseudomonas aeruginosa PAO1 resistance against a recently developed novel antibacterial zinc Schiff-base (ZSB) was investigated at the proteome level. After extended exposure to ZSB the passaged strain differed in tolerance against ZSB, with the adapted P. aeruginosa PAO1 exhibiting 1.6 times higher minimal inhibitory concentration. Using Two-dimensional Difference Gel Electrophoresis, the changes in the proteome of ZSB adapted P. aeruginosa PAO1 were examined by comparison with the non-adapted P. aeruginosa PAO1. The proteome of the adapted P. aeruginosa PAO1 strain differed significantly from the non-adapted in the abundance of two proteins when both strains were grown under stressing conditions. One protein could be identified as the outer membrane protein D that plays a role in uptake of basic amino acids as well as in carbapeneme resistance. The second protein has been identified as alkyl peroxide reductase subunit F. Our data indicated a slight increase in abundance of alkyl peroxide reductase F (AhpF) in the case of ZSB passaged P. aeruginosa PAO1. Higher abundance of Ahp has been discussed in the literature as a promoter of accelerated detoxification of benzene derivatives. The observed up-regulated AhpF thus appears to be connected to an increased tolerance against ZSB. Changes in the abundance of proteins connected to oxidative stress were also found after short-time exposure of P. aeruginosa PAO1 to the ZSB. Furthermore, adapted P. aeruginosa PAO1 showed increased tolerance against hydrogen peroxide and, in addition, showed accelerated degradation of ZSB, as determined by HPLC measurements. PMID:23869205

  20. Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa

    SciTech Connect

    Saiman, L.; Cacalano, G.; Prince, A. )

    1990-08-01

    Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

  1. Efficacy of VIP as Treatment for Bacteria-Induced Keratitis Against Multiple Pseudomonas aeruginosa Strains

    PubMed Central

    Carion, Thomas W.; McWhirter, Cody R.; Grewal, Daiyajot K.; Berger, Elizabeth A.

    2015-01-01

    Purpose Previous studies have demonstrated the efficacy of vasoactive intestinal peptide (VIP) treatment in regulating inflammation following bacterial keratitis induced by the P. aeruginosa strain 19660. However, in the current study we assessed whether disease outcome is specific to 19660 or if VIP treatment is effective against multiple P. aeruginosa strains. Methods B6 mice received daily IP injections of VIP from −1 through 5 days post injection (p.i.). Control mice were similarly injected with PBS. Corneal infection was induced using PA 19660, PAO1 or KEI 1025. Disease response was documented and bacterial plate counts and myeloperoxidase assays were performed. Expression of select inflammatory mediators as well as enzymes associated with lipid mediator production was assessed after VIP treatment. KEI 1025 was characterized by cytotoxicity and invasion assays and then confirmed for ExoS/ExoU expression. Results VIP treatment converted the susceptible response to resistant for the three P. aeruginosa strains tested. Disease response was significantly reduced with no corneal perforation. Anti-inflammatory mediators were enhanced after VIP treatment, while pro-inflammatory molecules were reduced compared to controls. Furthermore, VIP reduced inflammatory cell persistence in the cornea after infection with each of the P. aeruginosa strains. Conclusions VIP treatment is effective at ameliorating disease pathogenesis for multiple P. aeruginosa strains, both cytotoxic and invasive. This study is also the first to indicate a possible role for VIP regarding lipid mediator expression in the eye. In addition, the clinical isolate, KEI 1025, was characterized as an invasive strain. Overall, this study strengthens the preclinical development of VIP as a therapeutic agent for ocular infectious disease. PMID:26513498

  2. MrkD1P from Klebsiella pneumoniae Strain IA565 Allows for Coexistence with Pseudomonas aeruginosa and Protection from Protease-Mediated Biofilm Detachment

    PubMed Central

    Childers, Brandon M.; Van Laar, Tricia A.; You, Tao; Clegg, Steven

    2013-01-01

    Biofilm formation and persistence are essential components for the continued survival of pathogens inside the host and constitute a major contributor to the development of chronic wounds with resistance to antimicrobial compounds. Understanding these processes is crucial for control of biofilm-mediated disease. Though chronic wound infections are often polymicrobial in nature, much of the research on chronic wound-related microbes has focused on single-species models. Klebsiella pneumoniae and Pseudomonas aeruginosa are microbes that are often found together in wound isolates and are able to form stable in vitro biofilms, despite the antagonistic nature of P. aeruginosa with other organisms. Mutants of the K. pneumoniae strain IA565 lacking the plasmid-borne mrkD1P gene were less competitive than the wild type in an in vitro dual-species biofilm model with P. aeruginosa (PAO1). PAO1 spent medium inhibited the formation of biofilm of mrkD1P-deficient mutants and disrupted preestablished biofilms, with no effect on IA565 and no effect on the growth of the wild type or mutants. A screen using a two-allele PAO1 transposon library identified the LasB elastase as the secreted effector involved in biofilm disruption, and a purified version of the protein produced results similar to those with PAO1 spent medium. Various other proteases had a similar effect, suggesting that the disruption of the mrkD1P gene causes sensitivity to general proteolytic effects and indicating a role for MrkD1P in protection against host antibiofilm effectors. Our results suggest that MrkD1P allows for competition of K. pneumoniae with P. aeruginosa in a mixed-species biofilm and provides defense against microbial and host-derived proteases. PMID:23980108

  3. Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.

    PubMed Central

    Wolz, C; Hohloch, K; Ocaktan, A; Poole, K; Evans, R W; Rochel, N; Albrecht-Gary, A M; Abdallah, M A; Döring, G

    1994-01-01

    Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E. Images PMID:8063422

  4. LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants▿

    PubMed Central

    Steindler, Laura; Bertani, Iris; De Sordi, Luisa; Schwager, Stephan; Eberl, Leo; Venturi, Vittorio

    2009-01-01

    Pseudomonas aeruginosa possesses three quorum-sensing (QS) systems which are key in the expression of a large number of genes, including many virulence factors. Most studies of QS in P. aeruginosa have been performed in clinical isolates and have therefore focused on its role in pathogenicity. P. aeruginosa, however, is regarded as a ubiquitous organism capable of colonizing many different environments and also of establishing beneficial associations with plants. In this study we examined the role of the two N-acyl homoserine lactone systems known as RhlI/R and LasI/R in the environmental rice rhizosphere isolate P. aeruginosa PUPa3. Both the Rhl and Las systems are involved in the regulation of plant growth-promoting traits. The environmental P. aeruginosa PUPa3 is pathogenic in two nonmammalian infection models, and only the double las rhl mutants are attenuated for virulence. In fact it was established that the two QS systems are not hierarchically organized and that they are both important for the colonization of the rice rhizosphere. This is an in-depth genetic and molecular study of QS in an environmental P. aeruginosa strain and highlights several differences with QS regulation in the clinical isolate PAO1. PMID:19525275

  5. Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579

    PubMed Central

    Ryan Withers, T; Heath Damron, F; Yin, Yeshi; Yu, Hongwei D

    2013-01-01

    Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA108), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA108 confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA108, algW, algU, and rpoN encoding the alternative sigma factor σ54. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway. PMID:23533140

  6. Elastase Deficiency Phenotype of Pseudomonas aeruginosa Canine Otitis Externa Isolates

    PubMed Central

    Petermann, Shana R.; Doetkott, Curt; Rust, Lynn

    2001-01-01

    Pseudomonas aeruginosa veterinary isolates were assayed for elastase and total matrix protease activity. The elastase activity of canine ear isolates was much less than that of strain PAO1 and that of all other veterinary isolates (P < 0.0001). The results indicate that canine ear isolates have a distinct elastase phenotype. PMID:11329471

  7. Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature

    PubMed Central

    Penesyan, Anahit; Kumar, Sheemal S.; Kamath, Karthik; Shathili, Abdulrahman M.; Venkatakrishnan, Vignesh; Krisp, Christoph; Packer, Nicolle H.; Molloy, Mark P.; Paulsen, Ian T.

    2015-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such

  8. The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis.

    PubMed

    Gałczyńska, Katarzyna; Kurdziel, Krystyna; Adamus-Białek, Wioletta; Wąsik, Sławomir; Szary, Karol; Drabik, Marcin; Węgierek-Ciuk, Aneta; Lankoff, Anna; Arabski, Michał

    2015-01-01

    Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals. PMID:26645324

  9. Optimization and comparative characterization of neuraminidase activities from Pseudomonas aeruginosa with Klebsiella pneumoniae, Hep-2 cell, sheep kidney and rat liver lysosome

    PubMed Central

    Ghazaei, C; Ahmadi, M; Hosseini Jazani, N

    2010-01-01

    Background and Objectives The properties of neuraminidase produced by P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation. Materials and Methods The enzyme was isolated from concentrated culture supernatants of P. aeruginosa which was used in a sensitive fluorometric assay by using 2′-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate. Results Neuraminidase production in P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca+2, EDTA and NANA also decreased activity markedly. Conclusion The results revealed that the highest specific activity is for p. aeruginosa PAO1. PMID:22347548

  10. ZnuA and zinc homeostasis in Pseudomonas aeruginosa

    PubMed Central

    Pederick, Victoria G.; Eijkelkamp, Bart A.; Begg, Stephanie L.; Ween, Miranda P.; McAllister, Lauren J.; Paton, James C.; McDevitt, Christopher A.

    2015-01-01

    Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Central to the ability of P. aeruginosa to colonise both environmental and host niches is the acquisition of zinc. Here we show that P. aeruginosa PAO1 acquires zinc via an ATP-binding cassette (ABC) permease in which ZnuA is the high affinity, zinc-specific binding protein. Zinc uptake in Gram-negative organisms predominantly occurs via an ABC permease, and consistent with this expectation a P. aeruginosa ΔznuA mutant strain showed an ~60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. However, the effect of zinc limitation on the transcriptome of P. aeruginosa PAO1 revealed significant changes in gene expression that enable adaptation to low-zinc conditions. Genes significantly up-regulated included non-zinc-requiring paralogs of zinc-dependent proteins and a number of novel import pathways associated with zinc acquisition. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation. PMID:26290475

  11. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

    PubMed

    Choudhary, Geetika S; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  12. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  13. Cells of Escherichia coli are protected against severe chemical stress by co-habiting cell aggregates formed by Pseudomonas aeruginosa.

    PubMed

    Jagmann, Nina; Henke, Sebastian Franz; Philipp, Bodo

    2015-10-01

    Bacterial cells within biofilms and cell aggregates show increased resistance against chemical stress compared with suspended cells. It is not known whether bacteria that co-habit biofilms formed by other bacteria also acquire such resistance. This scenario was investigated in a proof-of-principle experiment with Pseudomonas aeruginosa strain PAO1 as cell aggregate-forming bacterium and Escherichia coli strain MG1655 as potential co-habiting bacterium equipped with an inducible bioluminescence system. Cell aggregation of strain PAO1 can be induced by the toxic detergent sodium dodecyl sulfate (SDS). In single cultures of strain MG1655, bioluminescence was inhibited by the protonophor carbonylcyanide-m-chlorophenylhydrazone (CCCP) but the cells were still viable. By applying CCCP and SDS together, cells of strain MG1655 lost their bioluminescence and viability indicating the importance of energy-dependent resistance mechanisms against SDS. In co-suspensions with strain PAO1, bioluminescence of strain MG1655 was sustained in the presence of SDS and CCCP. Image analysis showed that bioluminescent cells were located in cell aggregates formed by strain PAO1. Thus, cells of strain MG1655 that co-habited cell aggregates formed by strain PAO1 were protected against a severe chemical stress that was lethal to them in single cultures. Co-habiting could lead to increased survival of pathogens in clinical settings and could be employed in biotechnological applications involving toxic milieus. PMID:26066844

  14. Synergistic effect of membrane-active peptides polymyxin B and gramicidin S on multidrug-resistant strains and biofilms of Pseudomonas aeruginosa.

    PubMed

    Berditsch, Marina; Jäger, Thomas; Strempel, Nikola; Schwartz, Thomas; Overhage, Jörg; Ulrich, Anne S

    2015-09-01

    Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa. PMID:26077259

  15. A Drug-Repositioning Screening Identifies Pentetic Acid as a Potential Therapeutic Agent for Suppressing the Elastase-Mediated Virulence of Pseudomonas aeruginosa

    PubMed Central

    Gi, Mia; Jeong, Junhui; Lee, Keehoon; Lee, Kang-Mu; Toyofuku, Masanori; Yong, Dong Eun

    2014-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 μM DTPA. Supplementation with Zn2+ or Mn2+ ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection. PMID:25246397

  16. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis

    PubMed Central

    Silo-Suh, Laura; Suh, Sang-Jin; Sokol, Pamela A.; Ohman, Dennis E.

    2002-01-01

    A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease. Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection. However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection. Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50%. A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth in planta. FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis. In contrast, in a non-CF strain, PAO1, an algT mutation did not affect its virulence on alfalfa. Conversely, PAO1 virulence was reduced in a mucA mutant that overproduced alginate. These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection. These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of P. aeruginosa in CF. PMID:12426404

  17. A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus.

    PubMed

    Gi, Mia; Lee, Kang-Mu; Kim, Sang Cheol; Yoon, Joo-Heon; Yoon, Sang Sun; Choi, Jae Young

    2015-01-01

    Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection. PMID:26446565

  18. A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus

    PubMed Central

    Gi, Mia; Lee, Kang-Mu; Kim, Sang Cheol; Yoon, Joo-Heon; Yoon, Sang Sun; Choi, Jae Young

    2015-01-01

    Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection. PMID:26446565

  19. Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

    PubMed Central

    Tanaka, E; Kawamoto, S; Fukushima, J; Hamajima, K; Onishi, H; Miyagi, Y; Inami, S; Morihara, K; Okuda, K

    1991-01-01

    The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain. Images PMID

  20. Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.

    PubMed

    Hare, Nathan J; Soe, Cho Zin; Rose, Barbara; Harbour, Colin; Codd, Rachel; Manos, Jim; Cordwell, Stuart J

    2012-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further

  1. Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    PubMed Central

    Okkotsu, Yuta; Tieku, Prince; Fitzsimmons, Liam F.; Churchill, Mair E.

    2013-01-01

    AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ΔalgR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa. PMID:24097945

  2. In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients.

    PubMed

    Olszak, Tomasz; Zarnowiec, Paulina; Kaca, Wieslaw; Danis-Wlodarczyk, Katarzyna; Augustyniak, Daria; Drevinek, Pavel; de Soyza, Anthony; McClean, Siobhán; Drulis-Kawa, Zuzanna

    2015-07-01

    The goal of the study was to determine the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diversity between particular isolates and determined phage sensitivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PA were tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insensitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55-68 % of PA strains (72-86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bacterial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro assays, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations. PMID:25758956

  3. Alterations of OprD in Carbapenem-Intermediate and -Susceptible Strains of Pseudomonas aeruginosa Isolated from Patients with Bacteremia in a Spanish Multicenter Study

    PubMed Central

    Cabot, Gabriel; Rodríguez, Cristina; Roman, Elena; Tubau, Fe; Macia, María D.; Moya, Bartolomé; Zamorano, Laura; Suárez, Cristina; Peña, Carmen; Domínguez, María A.; Moncalián, Gabriel; Oliver, Antonio; Martínez-Martínez, Luis

    2012-01-01

    We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 μg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 μg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates (77%). Isolates were classified as OprD “full-length types” (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD “deficient types” (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging to OprD “deficient types” were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of only 0.06 to 4 μg/ml. PMID:22290967

  4. Reduction of PCN biosynthesis by NO in Pseudomonas aeruginosa.

    PubMed

    Gao, Lei; Zhang, Yuying; Wang, Yan; Qiao, Xinhua; Zi, Jing; Chen, Chang; Wan, Yi

    2016-08-01

    Pyocyanin (PCN), a virulence factor synthesized by Pseudomonas aeruginosa, plays an important role during clinical infections. There is no study of the effect of nitric oxide (NO) on PCN biosynthesis. Here, the effect of NO on PCN levels in Pseudomonas aeruginosa strain PAO1, a common reference strain, was tested. The results showed that the NO donor sodium nitroprusside (SNP) can significantly reduce PCN levels (82.5% reduction at 60μM SNP). Furthermore, the effect of endogenous NO on PCN was tested by constructing PAO1 nor (NO reductase gene) knockout mutants. Compared to the wild-type strain, the Δnor strain had a lower PCN (86% reduction in Δnor). To examine whether the results were universal with other P. aeruginosa strains, we collected 4 clinical strains from a hospital, tested their PCN levels after SNP treatment, and obtained similar results, i.e., PCN biosynthesis was inhibited by NO. These results suggest that NO treatment may be a new strategy to inhibit PCN biosynthesis and could provide novel insights into eliminating P. aeruginosa virulence as a clinical goal. PMID:26874276

  5. Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung

    PubMed Central

    Harmer, Christopher J.; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R.; Harbour, Colin; Triccas, James A.; Manos, Jim

    2015-01-01

    Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1. PMID:26252386

  6. Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

    PubMed

    Harmer, Christopher J; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R; Harbour, Colin; Triccas, James A; Manos, Jim

    2015-01-01

    Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1. PMID:26252386

  7. [New Virulent Bacteriophages Active against Multiresistant Pseudomonas aeruginosa Strains].

    PubMed

    Balarjishvili, N Sh; Kvachadze, L I; Kutateladze, M I; Meskhi, T Sh; Pataridze, T K; Berishvili, T A; Tevdoradze, E Sh

    2015-01-01

    The sensitivity of 512 newly isolated Pseudomonas aeruginosa clinical strains to six classes of anti-microbial preparations has been studied. Antibiotic-resistant strains were selected and genotyped. Three new virulent bacteriophages of the families Myoviridae and Podoviridae were isolated against these strains. The parameters of the intracellular phage development cycle were established, and the influence of inactivating factors (temperature, pH, and UV exposure) on phage viability was studied. The molecular weight of the phage genome was determined. Phage DNA restriction analysis and polyacrylamide gel electrophoresis in the presence of envelope protein SDS were carried out. The plating efficacy of phages on 28 genetically distant antibiotic-resistant P. aeruginosa strains was studied. It was established that 26 of them were lysed by phages with a high efficacy. The range of antibacterial action of the studied phages and their mixtures on 427 multi-drug-resistant clinical isolates was assessed. It is shown that including these phages in one multicomponent preparation enhanced their lytic activity. PMID:26859962

  8. Comparative sensitivity and resistance of some strains of Pseudomonas aeruginosa and Pseudomonas stutzeri to antibacterial agents

    PubMed Central

    Russell, A. D.; Mills, A. P.

    1974-01-01

    A comparison has been made of the sensitivities to various antibiotic and non-antibiotic substances of some strains of Pseudomonas aeruginosa and P. stutzeri, the latter including strains isolated from eye and other cosmetic products and from other sources. Whereas P. aeruginosa strains showed a high resistance to cetrimide and to benzalkonium chloride, the P. stutzeri strains were generally more sensitive to these and to chlorhexidine. The P. stutzeri strains were also more sensitive to the various antibiotics tested. The loss of the ability to transfer an R factor by two strains of P. aeruginosa caused no significant change in their drug sensitivity pattern. PMID:4369876

  9. Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons.

    PubMed

    Rojo-Bezares, Beatriz; Estepa, Vanesa; Cebollada, Rocío; de Toro, María; Somalo, Sergio; Seral, Cristina; Castillo, Francisco Javier; Torres, Carmen; Sáenz, Yolanda

    2014-05-01

    Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46. PMID:24594145

  10. Draft Genome Sequence of a Klebsiella pneumoniae Carbapenemase-Positive Sequence Type 111 Pseudomonas aeruginosa Strain

    PubMed Central

    Dotson, Gabrielle A.; Dekker, John P.; Palmore, Tara N.; Segre, Julia A.

    2016-01-01

    Here, we report the draft genome sequence of a sequence type 111 Pseudomonas aeruginosa strain isolated in 2014 from a patient at the NIH Clinical Center. This P. aeruginosa strain exhibits pan-drug resistance and harbors the blaKPC-2 gene, encoding the Klebsiella pneumoniae carbapenemase enzyme, on a plasmid. PMID:26868386

  11. Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

    PubMed Central

    Rivera, M; Bryan, L E; Hancock, R E; McGroarty, E J

    1988-01-01

    Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different. Images PMID:3123455

  12. A Novel Insight into Dehydroleucodine Mediated Attenuation of Pseudomonas aeruginosa Virulence Mechanism

    PubMed Central

    Mustafi, S.; Veisaga, M. L.; López, L. A.; Barbieri, M. A.

    2015-01-01

    Increasing resistance of Pseudomonas aeruginosa (P. aeruginosa) to conventional treatments demands the search for novel therapeutic strategies. In this study, the antimicrobial activity of dehydroleucodine (DhL), a sesquiterpene lactone obtained from Artemisia (A.) douglasiana, was screened against several pathogenic virulence effectors of P. aeruginosa. In vitro, minimum inhibitory concentration of DhL was determined against P. aeruginosa strains PAO1, PA103, PA14, and multidrug resistant clinical strain, CDN118. Results showed that DhL was active against each strain where PAO1 and PA103 showed higher susceptibility (MIC 0.48 mg/mL) as compared to PA14 (MIC 0.96 mg/mL) and CDN118 (MIC 0.98 mg/mL). Also, when PAO1 strain was grown in the presence of DhL (MIC50, 0.12 mg/mL), a delay in the generation time was noticed along with significant inhibition of secretory protease and elastase activities, interruption in biofilm attachment phase in a stationary culture, and a significant decline in Type III effector ExoS. At MIC50, DhL treatment increased the sensitivity of P. aeruginosa towards potent antibiotics. Furthermore, treatment of P. aeruginosa with DhL prevented toxin-induced apoptosis in macrophages. These observations suggest that DhL activity was at the bacterial transcriptional level. Hence, antimicrobial activity of DhL may serve as leads in the development of new anti-Pseudomonas pharmaceuticals. PMID:26640783

  13. The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

    PubMed

    Qaisar, Uzma; Kruczek, Cassandra J; Azeem, Muhammed; Javaid, Nasir; Colmer-Hamood, Jane A; Hamood, Abdul N

    2016-08-01

    Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope. PMID:27480638

  14. Temperature-Dependent Expression of phzM and Its Regulatory Genes lasI and ptsP in Rhizosphere Isolate Pseudomonas sp. Strain M18▿

    PubMed Central

    Huang, Jiaofang; Xu, Yuquan; Zhang, Hongyan; Li, Yaqian; Huang, Xianqing; Ren, Bin; Zhang, Xuehong

    2009-01-01

    Pseudomonas sp. strain M18, an effective biological control agent isolated from the melon rhizosphere, has a genetic background similar to that of the opportunistic human pathogen Pseudomonas aeruginosa PAO1. However, the predominant phenazine produced by strain M18 is phenazine-1-carboxylic acid (PCA) rather than pyocyanin (PYO); the quantitative ratio of PCA to PYO is 105 to 1 at 28°C in strain M18, while the ratio is 1 to 2 at 37°C in strain PAO1. We first provided evidence that the differential production of the two phenazines in strains M18 and PAO1 is related to the temperature-dependent and strain-specific expression patterns of phzM, a gene involved in the conversion of PCA to PYO. Transcriptional levels of phzM were measured by quantitative real-time PCR, and the activities of both transcriptional and translational phzM′-′lacZ fusions were determined in strains M18 and PAO1, respectively. Using lasI::Gm and ptsP::Gm inactivation M18 mutants, we further show that expression of the phzM gene is positively regulated by the quorum-sensing protein LasI and negatively regulated by the phosphoenolpyruvate phosphotransferase protein PtsP. Surprisingly, the lasI and ptsP regulatory genes were also expressed in a temperature-dependent and strain-specific manner. The differential production of the phenazines PCA and PYO by strains M18 and PAO1 may be a consequence of selective pressure imposed on P. aeruginosa PAO1 and its relative M18 in the two different niches over a long evolutionary process. PMID:19717631

  15. Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis.

    PubMed

    Pihl, Maria; Davies, Julia R; Chávez de Paz, Luis E; Svensäter, Gunnel

    2010-08-01

    Pseudomonas aeruginosa and Staphylococcus epidermidis are common opportunistic pathogens associated with medical device-related biofilm infections. 16S rRNA-FISH and confocal laser scanning microscopy were used to study these two bacteria in dual-species biofilms. Two of the four S. epidermidis strains used were shown to form biofilms more avidly on polymer surfaces than the other two strains. In dual-species biofilms, the presence of P. aeruginosa reduced biofilm formation by S. epidermidis, although different clinical isolates differed in their susceptibility to this effect. The most resistant isolate coexisted with P. aeruginosa for up to 18 h and was also resistant to the effects of the culture supernatant from P. aeruginosa biofilms, which caused dispersal from established biofilms of other S. epidermidis strains. Thus, different strains of S. epidermidis differed in their capacity to withstand the action of P. aeruginosa, with some being better equipped than others to coexist in biofilms with P. aeruginosa. Our data suggest that where S. epidermidis and P. aeruginosa are present on abiotic surfaces such as medical devices, S. epidermidis biofilm formation can be inhibited by P. aeruginosa through two mechanisms: disruption by extracellular products, possibly polysaccharides, and, in the later stages, by cell lysis. PMID:20528934

  16. Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

    PubMed Central

    Suh, Sang-Jin; Silo-Suh, Laura; Woods, Donald E.; Hassett, Daniel J.; West, Susan E. H.; Ohman, Dennis E.

    1999-01-01

    The sigma factor RpoS (ςS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the

  17. Marine-derived quorum-sensing inhibitory activities enhance the antibacterial efficacy of tobramycin against Pseudomonas aeruginosa.

    PubMed

    Busetti, Alessandro; Shaw, George; Megaw, Julianne; Gorman, Sean P; Maggs, Christine A; Gilmore, Brendan F

    2015-01-01

    Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (-63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (-33%). KS8 supernatant also caused a 0.97-log reduction (-89%) and a 2-log reduction (-99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments. PMID:25546516

  18. Marine-Derived Quorum-Sensing Inhibitory Activities Enhance the Antibacterial Efficacy of Tobramycin against Pseudomonas aeruginosa

    PubMed Central

    Busetti, Alessandro; Shaw, George; Megaw, Julianne; Gorman, Sean P.; Maggs, Christine A.; Gilmore, Brendan F.

    2014-01-01

    Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (−63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (−33%). KS8 supernatant also caused a 0.97-log reduction (−89%) and a 2-log reduction (−99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments. PMID:25546516

  19. Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response

    PubMed Central

    Bauman, Susanne J.; Kuehn, Meta J.

    2012-01-01

    Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation. PMID:16807039

  20. Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates.

    PubMed

    Conibear, Tim C R; Willcox, Mark D P; Flanagan, Judith L; Zhu, Hua

    2012-02-01

    Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system. PMID:21921113

  1. Antibiofilm activity of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 against Pseudomonas aeruginosa.

    PubMed

    Kim, Yong-Guy; Lee, Jin-Hyung; Kim, Chang-Jin; Lee, Jae-Chan; Ju, Yoon Jung; Cho, Moo Hwan; Lee, Jintae

    2012-12-01

    Members of the actinomycetes family are a rich source of bioactive compounds including diverse antibiotics. This study sought to identify novel and non-toxic biofilm inhibitors from the actinomycetes library for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. After the screening of 4104 actinomycetes strains, we found that the culture spent medium (1 %, v/v) of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 inhibited P. aeruginosa biofilm formation by 90 % without affecting the growth of planktonic P. aeruginosa cells, while the spent media enhanced the swarming motility of P. aeruginosa. Global transcriptome analyses revealed that the spent medium of Streptomyces sp. BFI 230 induced expression of phenazine, pyoverdine, pyochelin synthesis genes, and iron uptake genes in P. aeruginosa. The addition of exogenous iron restored the biofilm formation and swarming motility of P. aeruginosa in the presence of the spent medium of Streptomyces sp. BFI 230, which suggests that the Streptomyces sp. BFI 230 strain interfered iron acquisition in P. aeruginosa. Experiments on solvent extraction, heat treatment, and proteinase K treatment suggested that hydrophilic compound(s), possibly extracellular peptides or proteins from Streptomyces sp. BFI 230 cause the biofilm reduction of P. aeruginosa. Together, this study indicates that actinomycetes strains have an ability to control the biofilm of P. aeruginosa. PMID:22722911

  2. Pseudomonas aeruginosa inhibits the growth of Scedosporium aurantiacum, an opportunistic fungal pathogen isolated from the lungs of cystic fibrosis patients

    PubMed Central

    Kaur, Jashanpreet; Pethani, Bhavin P.; Kumar, Sheemal; Kim, Minkyoung; Sunna, Anwar; Kautto, Liisa; Penesyan, Anahit; Paulsen, Ian T.; Nevalainen, Helena

    2015-01-01

    The filamentous fungus Scedosporium aurantiacum and the bacterium Pseudomonas aeruginosa are opportunistic pathogens isolated from lungs of the cystic fibrosis (CF) patients. P. aeruginosa has been known to suppress the growth of a number of CF related fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. However, the interactions between P. aeruginosa and S. aurantiacum have not been investigated in depth. Hence we assessed the effect of P. aeruginosa reference strain PAO1 and two clinical isolates PASS1 and PASS2 on the growth of two clinical S. aurantiacum isolates WM 06.482 and WM 08.202 using solid plate assays and liquid cultures, in a synthetic medium mimicking the nutrient condition in the CF sputum. Solid plate assays showed a clear inhibition of growth of both S. aurantiacum strains when cultured with P. aeruginosa strains PASS1 and PAO1. The inhibitory effect was confirmed by confocal microscopy. In addition to using chemical fluorescent stains, strains tagged with yfp (P. aeruginosa PASS1) and mCherry (S. aurantiacum WM 06.482) were created to facilitate detailed microscopic observations on strain interaction. To our knowledge, this is the first study describing successful genetic transformation of S. aurantiacum. Inhibition of growth was observed only in co-cultures of P. aeruginosa and S. aurantiacum; the cell fractions obtained from independent bacterial monocultures failed to initiate a response against the fungus. In the liquid co-cultures, biofilm forming P. aeruginosa strains PASS1 and PAO1 displayed higher inhibition of fungal growth when compared to PASS2. No change was observed in the inhibition pattern when direct cell contact between the bacterial and fungal strains was prevented using a separation membrane suggesting the involvement of extracellular metabolites in the fungal inhibition. However, one of the most commonly described bacterial virulence factors, pyocyanin, had no effect against either of the S

  3. Antimicrobial susceptibility and molecular epidemiological analysis of clinical strains of Pseudomonas aeruginosa.

    PubMed

    Tsuchimochi, Noriko; Takuma, Takahiro; Shimono, Nobuyuki; Nagasaki, Yoji; Uchida, Yujiro; Harada, Mine

    2008-04-01

    Three hundred and seventy-one strains of Pseudomonas aeruginosa were isolated at the laboratory of Kyushu University Hospital in Japan from May 2002 through January 2003. Large proportions of isolated strains were resistant to carbapenems: 37.5% to imipenem, 21.3% to biapenem, and 18.3% to meropenem. A survey of injectable antibacterial agents used in our hospital during the corresponding period showed that carbapenems were most frequently used. Multidrug-resistant P. aeruginosa (MDRP) strains and metallo-beta-lactamase producing strains were isolated at frequencies of 1.6% (6 strains) and 0.81% (3 strains), respectively. By molecular epidemiological analysis, neither MDRP nor metallo-beta-lactamase producing strains were molecularly related, whereas some imipenem-resistant strains appeared to be epidemic strains, suggesting a possibility that they might spread by nosocomial infection. To control nosocomial infection, it is important to know a trend in drug-resistant P. aeruginosa and to prevent the spread of not only MDRP and metallo-beta-lactamase producing strains but also imipenem-resistant P. aeruginosa strains. PMID:18622671

  4. Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

    PubMed Central

    Chen, Y H; Hancock, R E; Mishell, R I

    1980-01-01

    Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens. Images Fig. 2 PMID:6769818

  5. Subtilase SprP exerts pleiotropic effects in Pseudomonas aeruginosa.

    PubMed

    Pelzer, Alexander; Polen, Tino; Funken, Horst; Rosenau, Frank; Wilhelm, Susanne; Bott, Michael; Jaeger, Karl-Erich

    2014-02-01

    The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ∆sprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms. PMID:24376018

  6. Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa

    PubMed Central

    Tin, San; Sakharkar, Kishore R.; Lim, Chu Sing; Sakharkar, Meena K.

    2009-01-01

    Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations. PMID:19173037

  7. Ambroxol interferes with Pseudomonas aeruginosa quorum sensing.

    PubMed

    Lu, Qi; Yu, Jialin; Yang, Xiqiang; Wang, Jiarong; Wang, Lijia; Lin, Yayin; Lin, Lihua

    2010-09-01

    The mucolytic agent ambroxol has been reported to interfere with the formation of Pseudomonas aeruginosa-derived biofilms in addition to reducing alginate production by undefined mechanisms. Since quorum sensing is a key regulator of virulence and biofilm formation, we examined the effects of ambroxol on P. aeruginosa PAO1 wild-type bacterial clearance rates, adhesion profiles and biofilm formation compared with the quorum sensing-deficient, double-mutant strains DeltalasR DeltarhlR and DeltalasI DeltarhlI. Data presented in this report demonstrated that ambroxol treatment reduced survival rates of the double-mutant strains compared with the wild-type strain in a dose-dependent manner even though the double-mutants had increased adhesion in the presence of ambroxol compared with the wild-type strain. The PAO1 wild-type strain produced a significantly thicker biofilm (21.64+/-0.57 microm) compared with the biofilms produced by the DeltalasR DeltarhlR (7.36+/-0.2 microm) and DeltalasI DeltarhlI (6.62+/-0.31 microm) isolates. Ambroxol treatment reduced biofilm thickness, increased areal porosity, and decreased the average diffusion distance and textual entropy of wild-type and double-mutant strains. However, compared with the double-mutant strains, the changes observed for the wild-type strain were more clearly defined. Finally, ambroxol exhibited significant antagonistic quorum-sensing properties, suggesting that it could be adapted for use clinically in the treatment of cystic fibrosis and to reduce biofilm formation and in the colonisation of indwelling devices. PMID:20580207

  8. Draft Genome Sequence of Quorum-Sensing and Quorum-Quenching Pseudomonas aeruginosa Strain MW3a

    PubMed Central

    Wong, Cheng Siang; Yin, Wai-Fong; Chan, Xin Yue

    2014-01-01

    Pseudomonas aeruginosa has a broad range of habitation, from aquatic environments to human lungs. The coexistence of quorum-sensing and quorum-quenching activities occurs in P. aeruginosa strain MW3a. In this work, we present the draft genome sequence of P. aeruginosa MW3a, an interesting bacterium isolated from a marine environment. PMID:24744329

  9. Identification of Novel Genomic Islands in Liverpool Epidemic Strain of Pseudomonas aeruginosa Using Segmentation and Clustering.

    PubMed

    Jani, Mehul; Mathee, Kalai; Azad, Rajeev K

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as "genomic islands (GIs)." To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, "GEMINI." GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa. PMID:27536294

  10. Identification of Novel Genomic Islands in Liverpool Epidemic Strain of Pseudomonas aeruginosa Using Segmentation and Clustering

    PubMed Central

    Jani, Mehul; Mathee, Kalai; Azad, Rajeev K.

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as “genomic islands (GIs).” To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, “GEMINI.” GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa. PMID:27536294

  11. Construction and Analysis of Photolyase Mutants of Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of Photoreactivation, Nucleotide Excision Repair, and Mutagenic DNA Repair to Cell Survival and Mutability following Exposure to UV-B Radiation

    PubMed Central

    Kim, Jae J.; Sundin, George W.

    2001-01-01

    Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2. Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m−2 followed by a recovery period under photoreactivating conditions. A phr uvrA mutant of P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m−2. Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 using the cloned phr gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant rulAB. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing rulAB indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which rulAB only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 × 10−5 and 2.1 × 10−3, respectively, following a UV-B dose of 1,550 J m−2. The construction and characterization of phr mutants in the present study will facilitate the

  12. Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody

    PubMed Central

    Behrouz, Bahador; Amirmozafari, Nour; Khoramabadi, Nima; Bahroudi, Mahboobeh; Legaee, Parisa; Mahdavi, Mehdi

    2016-01-01

    Background Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. Objectives As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin. Materials and Methods In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays. Results The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain. Conclusions The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity. PMID:27621933

  13. Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain.

    PubMed

    Ouidir, Tassadit; Jarnier, Frédérique; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2014-09-01

    Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes, signal transduction, and bacterial virulence. We characterized, for the first time, the extracellular phosphoproteins of the Pseudomonas aeruginosa PA14 strain. We identified 28 phosphoproteins (59 phosphosites) including enzymes, with various phosphorylation sites, known as potent secreted virulence factors in P. aeruginosa. The high phosphorylation level of these virulence factors might reflect a relationship between Ser/Thr/Tyr phosphorylation and virulence. PMID:24965220

  14. The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

    PubMed Central

    Essoh, Christiane; Blouin, Yann; Loukou, Guillaume; Cablanmian, Arsher; Lathro, Serge; Kutter, Elizabeth; Thien, Hoang Vu; Vergnaud, Gilles; Pourcel, Christine

    2013-01-01

    Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages. PMID:23637754

  15. Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

    PubMed

    Kamath, Karthik Shantharam; Pascovici, Dana; Penesyan, Anahit; Goel, Apurv; Venkatakrishnan, Vignesh; Paulsen, Ian T; Packer, Nicolle H; Molloy, Mark P

    2016-07-01

    Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens. PMID:27246823

  16. Evolution of metabolic divergence in Pseudomonas aeruginosa during long-term infection facilitates a proto-cooperative interspecies interaction.

    PubMed

    Frydenlund Michelsen, Charlotte; Hossein Khademi, Seyed Mohammad; Krogh Johansen, Helle; Ingmer, Hanne; Dorrestein, Pieter C; Jelsbak, Lars

    2016-06-01

    The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa-S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa-S. aureus relationship. PMID:26684729

  17. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    PubMed Central

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. PMID:27194047

  18. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa.

    PubMed

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. PMID:27194047

  19. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-05-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa.

  20. Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated Pseudomonas aeruginosa sp. Strain JQ-41.

    PubMed

    Song, Haihai; Zhou, Zhiren; Liu, Yuanxiu; Deng, Si; Xu, Heng

    2015-09-01

    A soil bacterium designated strain JQ-41, capable of growth on fenpropathrin as the sole carbon source and energy source, was isolated from a long-term pyrethroid insecticide-treated orchard. Based on the morphology, physio-biochemical characteristics, and 16S rDNA gene analysis, as well as the G+C content of the genomic DNA, the strain JQ-41 was identified as Pseudomonas aeruginosa. Up to 92.3% of 50 mg l(-1) fenpropathrin was degraded by P. aeruginosa strain at 30°C and pH 7 within 7 days. The kinetic parameters q max, K s, and K i were established to be 1.14 day(-1), 38.41 mg l(-1), and 137.67 mg l(-1), respectively, and the critical inhibitor concentration was determined to be 72.72 mg l(-1). Cell surface hydrophobicity of P. aeruginosa strain was enhanced during growth on fenpropathrin. Three metabolites from fenpropathrin degradation were identified by gas chromatography mass spectrometry, and then a possible degradation pathway was proposed. In addition, this isolate was also able to degrade a wide range of synthetic pyrethroid insecticides including cypermethrin, deltamethrin, bifenthrin, and cyhalothrin with the degradation process following the first-order kinetic model. Taken together, our results provide insights into the kinetics and mechanism of fenpropathrin degradation by P. aeruginosa strain and also highlight its promising potential in bioremediation of pyrethroid-contaminated environment. PMID:26068594

  1. Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from composts.

    PubMed

    Kaszab, Edit; Szoboszlay, Sándor; Dobolyi, Csaba; Háhn, Judit; Pék, Nikoletta; Kriszt, Balázs

    2011-01-01

    The aim of our work was to determine the presence of Pseudomonas aeruginosa in compost raw materials, immature and mature compost, and compost-treated soil. Twenty-five strains of P. aeruginosa were isolated from a raw material (plant straw), immature and mature compost and compost-treated soil samples. The strains were identified using the PCR method for the detection of species specific variable regions of 16S rDNA. Strains were examined for the presence of five different virulence-related gene sequences (exoA, exoU, exoT, exoS and exoY) and their antibiotic resistance profiles were determined. Based on our results, species P. aeruginosa can reach significant numbers (up to 10(6) MPN/g sample) during composting and 92.0% of the isolated strains carrying at least two gene sequences encoding toxic proteins. Various types of drug resistance were detected among compost originating strains, mainly against third generation Cephalosporins and Carbapenems. Six isolates were able to resist two different classes of antibiotics (third generation Cephalosporins and Carbapenems, wide spectrum Penicillins or Aminoglycosides, respectively). Based on our results, composts can be a source of P. aeruginosa and might be a concern to individuals susceptible to this opportunistic pathogen. PMID:20817443

  2. Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

    PubMed Central

    Facchini, Marcella; De Fino, Ida; Riva, Camilla; Bragonzi, Alessandra

    2014-01-01

    A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host. This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease. This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies. PMID:24686327

  3. Virulence factor expression patterns in Pseudomonas aeruginosa strains from infants with cystic fibrosis.

    PubMed

    Manos, J; Hu, H; Rose, B R; Wainwright, C E; Zablotska, I B; Cheney, J; Turnbull, L; Whitchurch, C B; Grimwood, K; Harmer, C; Anuj, S N; Harbour, C

    2013-12-01

    Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF). This study examines the role of organism-specific factors in the pathogenesis of very early P. aeruginosa infection in the CF airway. A total of 168 longitudinally collected P. aeruginosa isolates from children diagnosed with CF following newborn screening were genotyped by pulsed-field gel electrophoresis (PFGE) and phenotyped for 13 virulence factors. Ninety-two strains were identified. Associations between virulence factors and gender, exacerbation, persistence, timing of infection and infection site were assessed using multivariate regression analysis. Persistent strains showed significantly lower pyoverdine, rhamnolipid, haemolysin, total protease, and swimming and twitching motility than strains eradicated by aggressive antibiotic treatments. Initial strains had higher levels of virulence factors, and significantly higher phospholipase C, than subsequent genotypically different strains at initial isolation. Strains from males had significantly lower pyoverdine and swimming motility than females. Colony size was significantly smaller in strains isolated during exacerbation than those isolated during non-exacerbation periods. All virulence factors were higher and swimming motility significantly higher in strains from bronchoalveolar lavage (BAL) and oropharyngeal sites than BAL alone. Using unadjusted regression modelling, age at initial infection and age at isolation of a strain showed U-shaped profiles for most virulence factors. Among subsequent strains, longer time since initial infection meant lower levels of most virulence factors. This study provides new insight into virulence factors underpinning impaired airway clearance seen in CF infants, despite aggressive antibiotic therapy. This information will be important in the development of new strategies to reduce the impact of P. aeruginosa in CF. PMID:23832143

  4. Trigonella foenum-graceum (Seed) Extract Interferes with Quorum Sensing Regulated Traits and Biofilm Formation in the Strains of Pseudomonas aeruginosa and Aeromonas hydrophila

    PubMed Central

    Husain, Fohad Mabood; Ahmad, Iqbal; Khan, Mohd Shahnawaz; Al-Shabib, Nasser Abdulatif

    2015-01-01

    Trigonella foenum-graecum L. (Fenugreek) is an important plant of the Leguminosae family known to have medicinal properties. However, fraction based antiquorum sensing and antibiofilm activities have not been reported from this plant. In the present study T. foenum-graecum seed extract was sequentially fractionated and sub-MICs were tested for above activities. The methanol fraction of the extract demonstrated significant inhibition of AHL regulated virulence factors: protease, LasB elastase, pyocyanin production, chitinase, EPS, and swarming motility in Pseudomonas aeruginosa PAO1 and PAF79. Further, QS dependent virulence factor in the aquatic pathogen Aeromonas hydrophila WAF38 was also reduced. Application of T. foenum-graecum seed extract to PAO1, PAF79, and WAF38 decreased the biofilm forming abilities of the pathogens by significant levels. The extract also exhibited reduced AHL levels and subsequent downregulation of lasB gene. In vivo study showed an enhanced survival of PAO1-preinfected C. elegans after treatment with extract at 1 mg/mL. Further, the major compound detected by GC-MS, caffeine, reduced the production of QS regulated virulence factors and biofilm at 200 µg/mL concentration indicating its role in the activity of the methanol extract. The results of the present study reveal the potential anti-QS and antibiofilm property of T. foenum-graceum extract and caffeine. PMID:26000026

  5. Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

    PubMed Central

    Mugabe, Clement; Halwani, Majed; Azghani, Ali O.; Lafrenie, Robert M.; Omri, Abdelwahab

    2006-01-01

    Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of ≥32 versus ≤8 μg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ± 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ± 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ± 2.3 versus 24.2 ± 6.2 gold particles/bacterium, P ≤ 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells. PMID:16723560

  6. Mechanism of enhanced activity of liposome-entrapped aminoglycosides against resistant strains of Pseudomonas aeruginosa.

    PubMed

    Mugabe, Clement; Halwani, Majed; Azghani, Ali O; Lafrenie, Robert M; Omri, Abdelwahab

    2006-06-01

    Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of > or =32 versus < or =8 microg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% +/- 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% +/- 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 +/- 2.3 versus 24.2 +/- 6.2 gold particles/bacterium, P < or = 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells. PMID:16723560

  7. [Multicenter surveillance of Pseudomonas aeruginosa strains for antimicrobials in Aichi prefecture in 2009].

    PubMed

    Iguchi, Mitsutaka; Mochizuki, Mariko; Yagi, Tetsuya; Ookawa, Hironaga; Shimazaki, Yutaka; Ootsuka, Yumiko; Sato, Kazuya; Shiota, Arufumi; Wakiyama, Naoki; Nakamura, Atsushi; Kidono, Mariko; Hara, Yuki; Asai, Sachie; Kawashima, Makoto; Sakuragi, Kazuko; Asahi, Jitsuko; Murase, Hitoshi; Nishio, Mitsuru; Miyaki, Yuki; Funahashi, Keiji; Mouri, Tetsuo; Sugiura, Yasuyuki; Yamada, Takako; Kondo, Konomi; Sahara, Kaori; Sugaki, Yoshiko; Kawabata, Atsushi; Itou, Yumi; Yamamoto, Yu; Kinoshita, Keiko; Yamaguchi, Ikuo; Sasano, Masaaki; Inukai, Tomomi; Matsui, Natsuko; Kuramae, Hitoshi; Okugawa, Masaru; Kawai, Hiroki; Shibata, Motohiro; Inuzuka, Kazuhisa; Yamada, Atsuko; Koita, Isao; Suematsu, Hiroyuki; Sawamura, Haruki; Yamagishi, Yuka; Mikamo, Hiroshige

    2013-08-01

    We investigated the susceptibility to antimicrobials of 204 Pseudomonas aeruginosa strains isolated from 21 hospitals in Aichi prefecture from September to November 2009. MIC distributions of various antimicrobials were analyzed in terms of geographic region of isolation, patient status (outpatient or inpatient), and type of specimens that the strain was isolated from. The results were as follows. 1. Although more than 90% of strains were susceptible to all aminoglycosides and colistin, 80-90% of them were susceptible to beta-lactams and fluoroquinolones. MIC distributions of all antimicrobials measured were not significantly different between regions. 2. Only 1 strain (0.5%) was multi-drug resistant Pseudomonas aeruginosa (MDRP). Thirteen strains (6.4%) showed imipenem MIC > or = 16 microg/mL, and 16 strains (7.8%) showed ciprofloxacin MIC > or = 4 microg/mL. These strains tended to be more isolated from urine, respiratory tract specimens, or surgical specimens. 3. The MICs of tazobactam/piperacillin, panipenem, meropenem, doripenem, biapenem, sulbactam/cefoperazone, cefepime, and aztreonam were significantly higher in strains isolated from inpatients than in those from outpatients. MIC distributions of antimicrobials other than beta-lactams were not significantly different between situations where strains were isolated. 4. MIC distributions of piperacillin, all carbapenems, cefepime, gentamicin, and all fluoroquinolones were significantly different among samples from which strains were isolated. The strains isolated from blood showed lower MICs against all antimicrobials than those from other samples. No difference was found in MIC distributions when categorized according to bacteremic origin. The MICs were apparently elevated against beta-lactams, fluoroquinolones, and gentamicin in strains isolated from respiratory tract specimens, and against beta-lactams, and fluoroquinolones in strains isolated from urine. It was suggested that in P. aeruginosa surveillance

  8. Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.

    PubMed

    Kolpen, Mette; Appeldorff, Cecilie F; Brandt, Sarah; Mousavi, Nabi; Kragh, Kasper N; Aydogan, Sevtap; Uppal, Haleema A; Bjarnsholt, Thomas; Ciofu, Oana; Høiby, Niels; Jensen, Peter Ø

    2016-02-01

    Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose 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 (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions. PMID:26458402

  9. The clinical Pseudomonas fluorescens MFN1032 strain exerts a cytotoxic effect on epithelial intestinal cells and induces Interleukin-8 via the AP-1 signaling pathway

    PubMed Central

    2010-01-01

    Background Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response. Results Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-κB pathway. Conclusions The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients. PMID:20698984

  10. Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS

    PubMed Central

    Chandrasekaran, Subathra Devi; Vaithilingam, Mohanasrinivasan; Shanker, Ravi; Kumar, Sanjeev; Thiyur, Swathi; Babu, Vaishnavi; Selvakumar, Jemimah Naine; Prakash, Suyash

    2015-01-01

    Background: 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. Objectives: The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement. Materials and Methods: 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. Results: 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

  11. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

    PubMed

    Sun, Li-Wei; Jiang, Wen-Jing; Sato, Hiroaki; Kawachi, Masanobu; Lu, Xi-Wu

    2016-01-01

    Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS. PMID:27227555

  12. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI–TOF MS and Polygenetic Analysis

    PubMed Central

    Sun, Li-Wei; Jiang, Wen-Jing; Sato, Hiroaki; Kawachi, Masanobu; Lu, Xi-Wu

    2016-01-01

    Matrix-assisted laser desorption–ionization-time-of-flight mass spectrometry (MALDI–TOF MS) was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI–TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI–TOF MS. PMID:27227555

  13. Comparative Protein Expression in Different Strains of the Bloom-forming Cyanobacterium Microcystis aeruginosa*

    PubMed Central

    Alexova, Ralitza; Haynes, Paul A.; Ferrari, Belinda C.; Neilan, Brett A.

    2011-01-01

    Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported. PMID:21610102

  14. Photodynamic inactivation of antibiotic resistant strain of Pseudomonas aeruginosa in vivo

    NASA Astrophysics Data System (ADS)

    Hashimoto, M. C. E.; Toffoli, D. J.; Prates, R. A.; Courrol, Lilia C.; Ribeiro, M. S.

    2009-06-01

    Burns are frequently contamined by pathogenic microorganisms and the widespread occurrence of antibiotic resistant strains of Pseudomonas aeruginosa in hospitals is a matter of growing concern. Hypocrellin B (HB) is a new generation photosensitizer extracted from the fungus Hypocrella bambusae with absorption bands at 460, 546 and 584 nm. Lanthanide ions change the HB molecular structure and a red shift in the absorption band is observed as well as an increase in the singlet oxygen quantum yield. In this study, we report the use of HB:La+3 to kill resistant strain of P. aeruginosa infected burns. Burns were produced on the back of mice and wounds were infected subcutaneously with 1x109 cfu/mL of P. aeruginosa. Three-hours after inoculation, the animals were divided into 4 groups: control, HB:La+3, blue LED and HB:La+3+blue LED. PDT was performed using 10μM HB:La+3 and 500mW light-emitting diode (LED) emitting at λ=470nm+/-20nm during 120s. The animals of all groups were killed and the infected skin was removed for bacterial counting. Mice with photosensitizer alone, light alone or untreated infected wounds presented 1x108 cfu/g while mice PDT-treated showed a reduction of 2 logs compared to untreated control. These results suggest that HB:La+3 associated to blue LED is effective in diminishing antibiotic resistant strain P. aeruginosa in infected burns.

  15. Evaluating synergy between marbofloxacin and gentamicin in Pseudomonas aeruginosa strains isolated from dogs with otitis externa.

    PubMed

    Jerzsele, Ákos; Pásztiné-Gere, Erzsébet

    2015-03-01

    The aim of this study was to determine antimicrobial susceptibility of Pseudomonas aeruginosa strains to marbofloxacin and gentamicin, and investigate the possible synergistic, additive, indifferent or antagonistic effects between the two agents. P. aeruginosa strains can develop resistance quickly against certain antibiotics if used alone, thus the need emerges to find synergistic combinations. A total of 68 P. aeruginosa strains isolated from dogs were examined. In order to describe interactions between marbofloxacin and gentamicin the checkerboard microdilution method was utilized. The MICs (minimum inhibitory concentrations) for marbofloxacin and gentamicin were in the range 0.25-64 mg/L and 0.25-32 mg/L, respectively. The combination of marbofloxacin and gentamicin was more effective with a MIC range of 0.031-8 mg/L and a MIC90 of 1 mg/L, compared to 16 mg/L for marbofloxacin alone and 8 mg/L for gentamicin alone. The FIC (fractional inhibitory concentration) indices ranged from 0.0945 (pronounced synergy) to 1.0625 (indifference). Synergy between marbofloxacin and gentamicin was found in 33 isolates. The mean FIC index is 0.546, which represents a partial synergistic/additive effect close to the full synergy threshold. In vitro results indicate that marbofloxacin and gentamicin as partially synergistic agents may prove clinically useful in combination therapy against P. aeruginosa infections. Although marbofloxacin is not used in the human practice, the interactions between fluoroquinolones and aminoglycosides may have importance outside the veterinary field. PMID:25823453

  16. Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales.

    PubMed

    Scott, Fiona W; Pitt, Tyrone L

    2004-07-01

    Most past studies of cross-infection with Pseudomonas aeruginosa among cystic fibrosis (CF) patients in the UK suggest that it is a rare occurrence. However, two recent reports of highly transmissible strains in patients in regional centres in England (Liverpool and Manchester) have raised questions as to the extent of the problem and prompted a nationwide survey to establish the distribution of P. aeruginosa strain genotypes among these patients. Isolates of P. aeruginosa were requested from over 120 hospitals in England and Wales and a sample size of approximately 20% of the CF patient population in each centre was recommended. In total, 1225 isolates were received from 31 centres (range 1 to 330). Single patient isolates were typed by SpeI macrorestriction and PFGE. A panel of strains of the common genotypes including representatives of reported transmissible strains was assembled and further characterized by fluorescent amplified fragment length polymorphism (FAFLP) genotyping. At least 72% of all patients harboured strains with unique genotypes. Small clusters of related strains were evident in some centres, presumably indicating limited transmission of local strains. The most prevalent strain was indistinguishable from that previously described as the 'Liverpool' genotype, and accounted for approximately 11% of patient isolates from 15 centres in England and Wales. The second most common genotype (termed Midlands 1) was recovered from 86 patients in nine centres and the third genotype, which matched closely the PFGE profile of Clone C, a genotype originally described in Germany, was found in eight centres and was isolated from 15 patients. A fourth genotype, identical to the published Manchester strain, was found in three centres. FAFLP analysis revealed some microheterogeneity among strains of the Liverpool genotype but all isolates of this genotype were positive by PCR for a strain-specific marker. These data suggest that cross-infection with P. aeruginosa

  17. Effects of Chlorine Stress on Pseudomonas aeruginosa Biofilm and Analysis of Related Gene Expressions.

    PubMed

    Kekeç, Özge; Gökalsın, Barış; Karaltı, İskender; Kayhan, Figen Esin; Sesal, Nüzhet Cenk

    2016-08-01

    Chlorine is deployed worldwide to clean waters and prevent water-originated illnesses. However, chlorine has a limited disinfection capacity against biofilms. Microorganisms form biofilms to protect themselves from biological threats such as disinfectant chemicals. Pseudomonas aeruginosa is an opportunistic pathogen and its biofilm form attaches to surfaces, living buried into exopolysaccharides, can be present in all watery environments including tap water and drinking water. This research aimed to study the biofilm trigger mechanism of the opportunistic pathogen P. aeruginosa PAO1 strain, which is known to form biofilm in water supply systems and human body, under chlorine stress levels. In addition to biofilm staining, certain genes that are relevant to the stress condition were selected for gene expression analysis. The bacteria cultures were grown under chlorine stress with concentrations of 0.5, 0.7 and 1 mg/l. Six gene regions were determined related to biofilm and stress response: rpoS, bifA, migA, katB, soxR, and algC. Biofilm formation was analyzed by basic fuchsin staining, and gene expressions were quantified by quantitative real-time PCR. According to the results, highest biofilm production was observed in P. aeruginosa PAO1 wild strain under no stress conditions. Higher biofilm amounts were observed for bacteria under 0.5 and 0.7 mg/l chlorine stress compared to 1 mg/l chlorine stress. PMID:27146505

  18. Inhibition of Pseudomonas aeruginosa biofilm formation by 2,2’-bipyridyl, lipoic, kojic and picolinic acids

    PubMed Central

    Çevik, Kübra; Ulusoy, Seyhan

    2015-01-01

    Objective(s): The inhibitory effects of iron chelators, and FeCl3 chelation on biofilm formation and swarming motility were investigated against an opportunistic human pathogen Pseudomonas aeruginosa. Materials and Methods: The inhibitory activity of 2,2’-bipyridyl, lipoic acid, kojic acid and picolinic acid on biofilm formation of P. aeruginosa strain PAO1 and three clinical isolates (P. aeruginosa PAK01, P. aeruginosa PAK02 and P. aeruginosa PAK03) were investigated, based on crystal violet assay, and swarming motility test. Results: The kojic, lipoic and picolinic acid inhibited biofilm formation by 5-33% in all tested P. aeruginosa isolates. When chelated iron was added, biofilm inhibition rates were determined to be 39-57%. Among the tested chelators against P. aeruginosa, lipoic acid (84%) and kojic acid (68%) presented the highest inhibition of swarming motility. This is the first study to report the inhibitory effect of lipoic acid on biofilm formation and swarming motility of P. aeruginosa. Conclusion: It is considered that lipoic and picolinic acids can serve as alternatives for the treatment of the P. aeruginosa infections by inhibiting biofilm formation. PMID:26557964

  19. Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Bedi, Brahmchetna; Yuan, Zhihong; Joo, Myungsoo; Zughaier, Susu M; Goldberg, Joanna B; Arbiser, Jack L; Hart, C Michael; Sadikot, Ruxana T

    2016-07-01

    The pathogenic profile of Pseudomonas aeruginosa is related to its ability to secrete a variety of virulence factors. Quorum sensing (QS) is a mechanism wherein small diffusible molecules, specifically acyl-homoserine lactones, are produced by P. aeruginosa to promote virulence. We show here that macrophage clearance of P. aeruginosa (PAO1) is enhanced by activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Macrophages treated with a PPARγ agonist (pioglitazone) showed enhanced phagocytosis and bacterial killing of PAO1. It is known that PAO1 QS molecules are inactivated by PON-2. QS molecules are also known to inhibit activation of PPARγ by competitively binding PPARγ receptors. In accord with this observation, we found that infection of macrophages with PAO1 inhibited expression of PPARγ and PON-2. Mechanistically, we show that PPARγ induces macrophage paraoxonase 2 (PON-2), an enzyme that degrades QS molecules produced by P. aeruginosa Gene silencing studies confirmed that enhanced clearance of PAO1 in macrophages by PPARγ is PON-2 dependent. Further, we show that PPARγ agonists also enhance clearance of P. aeruginosa from lungs of mice infected with PAO1. Together, these data demonstrate that P. aeruginosa impairs the ability of host cells to mount an immune response by inhibiting PPARγ through secretion of QS molecules. These studies define a novel mechanism by which PPARγ contributes to the host immunoprotective effects during bacterial infection and suggest a role for PPARγ immunotherapy for P. aeruginosa infections. PMID:27091928

  20. Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients

    PubMed Central

    Al-Aloul, M; Crawley, J; Winstanley, C; Hart, C; Ledson, M; Walshaw, M

    2004-01-01

    Background: Chronic pulmonary infection with transmissible Pseudomonas aeruginosa strains in individuals with cystic fibrosis (CF) has been reported, raising issues of cross infection and patient segregation. The first such strain to be described (the Liverpool epidemic strain, LES) is now widespread in many UK CF centres. However, whether such infection carries a worse prognosis is unknown. To address this, the clinical course of a group of CF patients chronically infected by LES was compared with that in patients harbouring unique strains. Methods: Using P aeruginosa strain genotyping, two cohorts of CF patients attending the Liverpool CF service were identified who were LES positive or negative in 1998 and remained so until 2002. From these, two groups of 12 patients were matched in 1998 for age, spirometric parameters, and nutritional state and their clinical course was followed for 5 years. Patients chronically infected with Burkholderia cepacia were excluded. Results: Patients chronically infected with LES had a greater annual loss of lung function than those not chronically infected by LES (mean difference between groups -4.4% (95% CI -8.1 to -0.9; p<0.02)), and by 2002 their percentage predicted forced expiratory volume in 1 second (FEV1) was worse (mean 65.0% v 82.6%, p<0.03). Their nutritional state also deteriorated over the study period (mean difference between groups in body mass index -0.7 (95% CI -1.2 to -0.2; p<0.01)), such that by 2002 they were malnourished compared with LES negative patients (mean BMI 19.4 v 22.7, p<0.02). Conclusions: Chronic infection with the Liverpool epidemic P aeruginosa strain in CF patients confers a worse prognosis than infection with unique strains alone, confirming the need for patient segregation. Since this strain is common in many CF units, strain identification in all CF centres is essential. This can only be carried out using genomic typing methods. PMID:15047956

  1. Outbreak of equine endometritis caused by a genotypically identical strain of Pseudomonas aeruginosa.

    PubMed

    Allen, Joanne L; Begg, Angela P; Browning, Glenn F

    2011-11-01

    Pseudomonas aeruginosa is an opportunistic pathogen that has been recognized as a cause of endometritis in mares. Pulsed field gel electrophoresis was used to characterize and compare isolates of P. aeruginosa from an outbreak of endometritis and unrelated isolates collected at the same time as the outbreak. The restriction endonuclease digestion patterns and antimicrobial resistance profiles of all outbreak isolates were identical. Therefore, a single strain of P. aeruginosa was responsible for the cases of endometritis. The unrelated isolates could be distinguished from the outbreak strain using the techniques outlined in the present study. The results establish that this pathogen was not venereally transmitted between all the horses from which it was isolated, but rather must have been disseminated, at least initially, from a contaminated water source. Once the water used to clean the mares and stallions was replaced, there were no further reports of endometritis caused by this organism on the affected stud. Furthermore, the fertility of the stallions was not affected, in spite of persistent carriage for 1 to 2 months. The current study has shown that the use of pulsed field gel electrophoresis has considerable value in epidemiological investigations of equine urogenital tract infections with P. aeruginosa. PMID:22362810

  2. [Incidence of alginate-coding gene in carbapenem-resistant Pseudomonas aeruginosa strains].

    PubMed

    Bogiel, Tomasz; Kwiecińska-Piróg, Joanna; Kozuszko, Sylwia; Gospodarek, Eugenia

    2011-01-01

    Pseudomonas aeruginosa rods are one of the most common isolated opportunistic nosocomial pathogens. Strains usually are capable to secret a capsule-like polysaccharide called alginate important for evasion of host defenses, especially during chronic pulmonary disease of patients with cystic fibrosis. Most genes for alginate biosynthesis and lysis are encoded by the operon. The aim of our study was to evaluate the incidence of algD sequence, generally use for alginate-coding gene detection, in 120 P. aeruginosa strains resistant to carbapenems. All isolates were obtained in the Department of Clinical Microbiology University Hospital no. 1 of dr A. Jurasz Collegium Medicum of L. Rydygier in Bydgoszcz Nicolaus Copernicus University in Toruń. Examined strains demonstrated resistance to carbenicillin (90,0%), ticarcillin (89,2%) and ticarcillin clavulanate (86,7%). All strains were susceptible to colistin. The majority of examined strains was susceptible to ceftazidime and cefepime (40,8% each) and norfloxacin (37,5%). Presence of algD gene - noted in 112 (93,3%) strains proves that not every strain is capable to produce alginate. It was also found out that differences in algD genes incidence in case of different clinical material that strains were isolated from were not statistically important. PMID:22184909

  3. Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods.

    PubMed

    Kalai Blagui, S; Achour, W; Bejaoui, M; Abdeladhim, A; Ben Hassen, A

    2009-05-01

    Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain. PMID:18456431

  4. Anticandidal activity of medicinal plants and Pseudomonas aeruginosa strains of clinical specimens.

    PubMed

    Bora, Limpon

    2016-04-01

    This study was designed to investigate the in vitro anticandidal activity of some medicinal plants and Pseudomonas aeruginosa strains against Candida species. The antifungal activity of methanolic extracts of five medicinal plants, namely, Cinnamomum porrectum, Lippia nudiflora, Cestrum nocturnum, Trachyspermum ammi, and Sida carpinifolia were studied. The medicinal characteristics of these plants were compared with commercially used antibiotics. The antimicrobial assay was done by agar well diffusion and the broth dilution method. Among the plants used, T. ammi and C. nocturnum were found to be more potent than the others. Twenty P. aeruginosa strains were isolated from various clinical specimens. The total inhibitions obtained were found to be 47%, 38%, and 36% in blood agar, whereas in Sabouraud dextrose agar (SDA) the inhibitions were 57%, 48%, and 37%, respectively. PMID:25592881

  5. Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are toxic to Lucilia sericata maggots

    PubMed Central

    Andersen, A. S.; Joergensen, B.; Bjarnsholt, T.; Johansen, H.; Karlsmark, T.; Givskov, M.; Krogfelt, K. A.

    2010-01-01

    Maggot debridement therapy (MDT) is widely used for debridement of chronic infected wounds; however, for wounds harbouring specific bacteria limited effect or failure of the treatment has been described. Here we studied the survival of Lucilia sericata maggots encountering Pseudomonas aeruginosa PAO1 in a simple assay with emphasis on the quorum-sensing (QS)-regulated virulence. The maggots were challenged with GFP-tagged P. aeruginosa wild-type (WT) PAO1 and a GFP-tagged P. aeruginosa ΔlasR rhlR (ΔRR) QS-deficient mutant in different concentrations. Maggots were killed in the presence of WT PAO1 whereas the challenge with the QS mutant showed a survival reduction of ∼25 % compared to negative controls. Furthermore, bacterial intake by the maggots was lower in the presence of WT PAO1 compared to the PAO1 ΔRR mutant. Maggot excretions/secretions (ES) were assayed for the presence of QS inhibitors; only high doses of ES showed inhibition of QS in P. aeruginosa. Thus P. aeruginosa was shown to be toxic to L. sericata maggots. This, coupled to the preferential feeding by the maggots and reduced ingestion of P. aeruginosa, could explain MDT failure in wounds colonized by P. aeruginosa. Wounds heavily colonized with P. aeruginosa should be a counterindication for MDT unless used in combination with a pre-treatment with other topical therapeutics targeting P. aeruginosa. PMID:19892758

  6. Draft Genome Sequence of a Clinically Isolated Extensively Drug-Resistant Pseudomonas aeruginosa Strain

    PubMed Central

    Manivannan, Bhavani; Mahalingam, Niranjana; Jadhao, Sudhir; Mishra, Amrita; Nilawe, Pravin

    2016-01-01

    We present the draft genome assembly of an extensively drug-resistant (XDR) Pseudomonas aeruginosa strain isolated from a patient with a history of genito urinary tuberculosis. The draft genome is 7,022,546 bp with a G+C content of 65.48%. It carries 7 phage genomes, genes for quorum sensing, biofilm formation, virulence, and antibiotic resistance. PMID:27013045

  7. Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

    PubMed Central

    Rakhimova, Elza; Munder, Antje; Wiehlmann, Lutz; Bredenbruch, Florian; Tümmler, Burkhard

    2008-01-01

    Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild. PMID:18301762

  8. Bactericidal activities of cathelicidin LL-37 and select cationic lipids against the hypervirulent Pseudomonas aeruginosa strain LESB58.

    PubMed

    Wnorowska, Urszula; Niemirowicz, Katarzyna; Myint, Melissa; Diamond, Scott L; Wróblewska, Marta; Savage, Paul B; Janmey, Paul A; Bucki, Robert

    2015-07-01

    Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains. PMID:25870055

  9. Bactericidal Activities of Cathelicidin LL-37 and Select Cationic Lipids against the Hypervirulent Pseudomonas aeruginosa Strain LESB58

    PubMed Central

    Wnorowska, Urszula; Niemirowicz, Katarzyna; Myint, Melissa; Diamond, Scott L.; Wróblewska, Marta; Savage, Paul B.; Janmey, Paul A.

    2015-01-01

    Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains. PMID:25870055

  10. Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Niewerth, Heiko

    2014-01-01

    A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule. PMID:25239889

  11. Mechanisms of plant growth promotion and disease suppression by Pseudomonas aeruginosa strain 2apa.

    PubMed

    Hariprasad, P; Chandrashekar, S; Singh, S Brijesh; Niranjana, S R

    2014-08-01

    A new Pseudomonas strain, designated as 2apa was isolated from tomato rhizosphere and identified as a member of species Pseudomonas aeruginosa based on its morphology, conventional, biochemical, cell wall fatty acid methyl ester analysis, and 16S rRNA gene sequence analysis. The strain 2apa was positive for root colonization, indole acetic acid (IAA), salicylic acid and siderophore production and inhibited the growth of wide range of microorganisms. Antimicrobial substances produced by this strain with further purification and structure elucidation proved to be phenazine. Under laboratory and greenhouse conditions the strain promoted plant growth and suppressed a wide range of foliar and root pathogens in tomato. The protection offered by strain 2apa to foliar pathogens is considered as induced systemic resistance and was further confirmed by enhanced accumulation of phenolics, elicitation of lipoxygenas activity, and jasmonic acid levels. The broad-spectrum antimicrobial and induced systemic resistance exhibiting strain P. aeruginosa 2apa can be used as an effective biological control candidate against devastating fungal and bacterial pathogens, which attack both root and foliar portions of tomato plant. Production of other functional traits such as IAA and siderophore may enhance its potential as biofertilizer. PMID:23681707

  12. Complete Genome Sequence of the Multiresistant Taxonomic Outlier Pseudomonas aeruginosa PA7

    PubMed Central

    Roy, Paul H.; Tetu, Sasha G.; Larouche, André; Elbourne, Liam; Tremblay, Simon; Ren, Qinghu; Dodson, Robert; Harkins, Derek; Shay, Ryan; Watkins, Kisha; Mahamoud, Yasmin; Paulsen, Ian T.

    2010-01-01

    Pseudomonas aeruginosa PA7 is a non-respiratory human isolate from Argentina that is multiresistant to antibiotics. We first sequenced gyrA, gyrB, parC, parE, ampC, ampR, and several housekeeping genes and found that PA7 is a taxonomic outlier. We report here the complete sequence of the 6,588,339 bp genome, which has only about 95% overall identity to other strains. PA7 has multiple novel genomic islands and a total of 51 occupied regions of genomic plasticity. These islands include antibiotic resistance genes, parts of transposons, prophages, and a pKLC102-related island. Several PA7 genes not present in PAO1 or PA14 are putative orthologues of other Pseudomonas spp. and Ralstonia spp. genes. PA7 appears to be closely related to the known taxonomic outlier DSM1128 (ATCC9027). PA7 lacks several virulence factors, notably the entire TTSS region corresponding to PA1690-PA1725 of PAO1. It has neither exoS nor exoU and lacks toxA, exoT, and exoY. PA7 is serotype O12 and pyoverdin type II. Preliminary proteomic studies indicate numerous differences with PAO1, some of which are probably a consequence of a frameshift mutation in the mvfR quorum sensing regulatory gene. PMID:20107499

  13. Selection of an Antibiotic-Hypersusceptible Mutant of Pseudomonas aeruginosa: Identification of the GlmR Transcriptional Regulator

    PubMed Central

    Ramos-Aires, Julio; Plésiat, Patrick; Kocjancic-Curty, Lasta; Köhler, Thilo

    2004-01-01

    Tn501 random mutagenesis was applied to the Pseudomonas aeruginosa wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents. One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, β-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides. Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells. The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures. The insertion site of the Tn501 transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors. The gene, which we called glmR, is located between the glmS (PA5549) and glmU (PA5552) homologues of E. coli, responsible for the synthesis of UDP-N-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan. We showed that GlmR represses the transcription of the adjacent glmS homologue (PA5549) in P. aeruginosa, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis. To our knowledge GlmR is the first regulator in P. aeruginosa that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents. PMID:14982774

  14. Quantification of Pseudomonas aeruginosa hydrogen cyanide production by a polarographic approach.

    PubMed

    Blier, Anne-Sophie; Vieillard, Julien; Gerault, Eloïse; Dagorn, Audrey; Varacavoudin, Tony; Le Derf, Franck; Orange, Nicole; Feuilloley, Marc; Lesouhaitier, Olivier

    2012-07-01

    Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used. PMID:22537820

  15. Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis

    PubMed Central

    Tai, Anna Sze; Bell, Scott Cameron; Kidd, Timothy James; Trembizki, Ella; Buckley, Cameron; Ramsay, Kay Annette; David, Michael; Wainwright, Claire Elizabeth; Grimwood, Keith; Whiley, David Mark

    2015-01-01

    In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001–2002 and 2007–2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the ‘M3L7’ subtype at this centre during 2007–2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007–2009 than 2001–2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1–13.5 and LasR: OR = 2.5; 95%CI: 1.3–5.0). Surveillance at the adult centre in 2007–2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9

  16. Genotypic Diversity within a Single Pseudomonas aeruginosa Strain Commonly Shared by Australian Patients with Cystic Fibrosis.

    PubMed

    Tai, Anna Sze; Bell, Scott Cameron; Kidd, Timothy James; Trembizki, Ella; Buckley, Cameron; Ramsay, Kay Annette; David, Michael; Wainwright, Claire Elizabeth; Grimwood, Keith; Whiley, David Mark

    2015-01-01

    In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001-2002 and 2007-2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the 'M3L7' subtype at this centre during 2007-2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007-2009 than 2001-2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1-13.5 and LasR: OR = 2.5; 95%CI: 1.3-5.0). Surveillance at the adult centre in 2007-2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2-39.2) and

  17. Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa.

    PubMed

    Latino, Libera; Midoux, Cédric; Hauck, Yolande; Vergnaud, Gilles; Pourcel, Christine

    2016-05-01

    Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10- 5 for single phage infection and 10- 6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure. PMID:26921273

  18. Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress.

    PubMed

    Romsang, Adisak; Atichartpongkul, Sopapan; Trinachartvanit, Wachareeporn; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

    2013-08-01

    Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive. PMID:23687271

  19. Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation.

    PubMed

    McElroy, Kerensa E; Hui, Janice G K; Woo, Jerry K K; Luk, Alison W S; Webb, Jeremy S; Kjelleberg, Staffan; Rice, Scott A; Thomas, Torsten

    2014-04-01

    Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10(-3) substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern short-term, within-population bacterial diversification. PMID:24706926

  20. Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen.

    PubMed

    Imperi, Francesco; Ciccosanti, Fabiola; Perdomo, Ariel Basulto; Tiburzi, Federica; Mancone, Carmine; Alonzi, Tonino; Ascenzi, Paolo; Piacentini, Mauro; Visca, Paolo; Fimia, Gian Maria

    2009-04-01

    The Gram-negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high-throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild-type P. aeruginosa strain PAO1 by 2-DE and MALDI-TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell-envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments. PMID:19333994

  1. Cloning, expression and purification of penicillin-binding protein 3 from Pseudomonas aeruginosa CMCC 10104.

    PubMed

    An, Yan Dong; Du, Qi Zhen; Tong, Li Yan; Yu, Zhao Wu; Gong, Xing Wen

    2015-06-01

    Penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa is the primary target of β-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in Escherichia coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104. PMID:25514204

  2. Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

    PubMed Central

    Winteler, H V; Schneidinger, B; Jaeger, K E; Haas, D

    1996-01-01

    The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase. PMID:8795231

  3. Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms.

    PubMed

    Pezzoni, Magdalena; Pizarro, Ramón A; Costa, Cristina S

    2014-02-01

    One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA(+) strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment. PMID:24491420

  4. Draft genome sequence of blaVeb-1, blaoxa-10 producing multi-drug resistant (MDR) Pseudomonas aeruginosa strain VRFPA09 recovered from bloodstream infection.

    PubMed

    Murugan, Nandagopal; Malathi, Jambulingam; Umashankar, Vetrivel; Madhavan, Hajib NarahariRao

    2015-01-01

    Pseudomonas aeruginosa (P. aeruginosa) bacteremia causes significant mortality rate due to emergence of multidrug resistant (MDR) nosocomial infections. We report the draft genome sequence of P. aeruginosa strain VRFPA09, a human bloodstream isolate, phenotypically proven as MDR strain. Whole genome sequencing on VRFPA09, deciphered betalactamase encoding blav(eb-1) and bla(OXA-10) genes and multiple drug resistance, virulence factor encoding genes. PMID:26413042

  5. Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment ▿

    PubMed Central

    Macià, María D.; Pérez, José L.; Molin, Soeren; Oliver, Antonio

    2011-01-01

    Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms. PMID:21859941

  6. Microcyclamide Biosynthesis in Two Strains of Microcystis aeruginosa: from Structure to Genes and Vice Versa▿ †

    PubMed Central

    Ziemert, Nadine; Ishida, Keishi; Quillardet, Philippe; Bouchier, Christiane; Hertweck, Christian; de Marsac, Nicole Tandeau; Dittmann, Elke

    2008-01-01

    Comparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacterium Microcystis aeruginosa NIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain, M. aeruginosa PCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria. PMID:18245249

  7. Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability

    PubMed Central

    Illakkiam, Devaraj; Shankar, Manoharan; Ponraj, Paramasivan; Rajendhran, Jeyaprakash

    2014-01-01

    Pseudomonas aeruginosa PGPR2 is a mung bean rhizosphere strain that produces secondary metabolites and hydrolytic enzymes contributing to excellent antifungal activity against Macrophomina phaseolina, one of the prevalent fungal pathogens of mung bean. Genome sequencing was performed using the Ion Torrent Personal Genome Machine generating 1,354,732 reads (6,772,433 sequenced bases) achieving ~25-fold coverage of the genome. Reference genome assembly using MIRA 3.4.0 yielded 198 contigs. The draft genome of PGPR2 encoded 6803 open reading frames, of which 5314 were genes with predicted functions, 1489 were genes of known functions, and 80 were RNA-coding genes. Strain specific and core genes of P. aeruginosa PGPR2 that are relevant to rhizospheric habitat were identified by pangenome analysis. Genes involved in plant growth promoting function such as synthesis of ACC deaminase, indole-3-acetic acid, trehalose, mineral scavenging siderophores, hydrogen cyanide, chitinases, acyl homoserine lactones, acetoin, 2,3-butanediol, and phytases were identified. In addition, niche-specific genes such as phosphate solubilising 3-phytase, adhesins, pathway-specific transcriptional regulators, a diguanylate cyclase involved in cellulose synthesis, a receptor for ferrienterochelin, a DEAD/DEAH-box helicase involved in stress tolerance, chemotaxis/motility determinants, an HtpX protease, and enzymes involved in the production of a chromanone derivative with potent antifungal activity were identified. PMID:25184130

  8. Deciphering the metabolic capabilities of a lipase producing Pseudomonas aeruginosa SL-72 strain.

    PubMed

    Verma, Shikha; Prasanna, Radha; Saxena, Jyoti; Sharma, Vinay; Nain, Lata

    2012-11-01

    Pseudomonads have been reported for their metabolic, nutritional and ecological versatility, which motivated us to prospect the metabolic profile of a lipolytic strain Pseudomonas aeruginosa SL-72. The strain SL-72 was found to produce high levels of lipase and pectinase (1,555.62 IU/mL and 1,490.33 IU/mL, respectively), esterase and amylase, besides low levels of xylanase, proteinase and cellulase. The strain also tested positive for different plant growth-promoting traits-production of ammonia, hydrogen cyanide, siderophores, phosphate solubilization, nitrate reduction and antifungal activity. The high levels of activity of aryl sulphatase, alkaline phosphatase and fluorescein diacetate hydrolase makes it a useful strain for enhanced nutrient cycling in soil. The strain SL-72 produced rhamnolipids, a biosurfactant and its production was enhanced when starch was used as carbon source (0.256 g/L) and utilized polycyclic hydrocarbon compounds viz. anthracene, phenanthrene, pyrene, fluorene and its mixture. The multifaceted nature of the culture illustrates its promise in bioremediation, industry, besides its use as an inoculant. PMID:22661061

  9. Phenotypic and Genotypic Comparison of Epidemic and Non-Epidemic Strains of Pseudomonas aeruginosa from Individuals with Cystic Fibrosis

    PubMed Central

    Duong, Jessica; Booth, Sean C.; McCartney, Nathan K.; Rabin, Harvey R.; Parkins, Michael D.; Storey, Douglas G.

    2015-01-01

    Epidemic strains of Pseudomonas aeruginosa have been found worldwide among the cystic fibrosis (CF) patient population. Using pulse-field gel electrophoresis, the Prairie Epidemic Strain (PES) has recently been found in one-third of patients attending the Calgary Adult CF Clinic in Canada. Using multi-locus sequence typing, PES isolates from unrelated patients were found to consistently have ST192. Though most patients acquired PES prior to enrolling in the clinic, some patients were observed to experience strain replacement upon transitioning to the clinic whereby local non-epidemic P. aeruginosa isolates were displaced by PES. Here we genotypically and phenotypically compared PES to other P. aeruginosa epidemic strains (OES) found around the world as well as local non-epidemic CF P. aeruginosa isolates in order to characterize PES. Since some epidemic strains are associated with worse clinical outcomes, we assessed the pathogenic potential of PES to determine if these isolates are virulent, shared properties with OES, and if its phenotypic properties may offer a competitive advantage in displacing local non-epidemic isolates during strain replacement. As such, we conducted a comparative analysis using fourteen phenotypic traits, including virulence factor production, biofilm formation, planktonic growth, mucoidy, and antibiotic susceptibility to characterize PES, OES, and local non-epidemic isolates. We observed that PES and OES could be differentiated from local non-epidemic isolates based on biofilm growth with PES isolates being more mucoid. Pairwise comparisons indicated that PES produced significantly higher levels of proteases and formed better biofilms than OES but were more susceptible to antibiotic treatment. Amongst five patients experiencing strain replacement, we found that super-infecting PES produced lower levels of proteases and elastases but were more resistant to antibiotics compared to the displaced non-epidemic isolates. This comparative

  10. Antibiotic Susceptibility Patterns and Molecular Epidemiology of Metallo-β-Lactamase Producing Pseudomonas Aeruginosa Strains Isolated From Burn Patients

    PubMed Central

    Japoni, Aziz; Anvarinejad, Mojtaba; Farshad, Shohreh; Giammanco, Giovanni M; Rafaatpour, Noroddin; Alipour, Ebrahim

    2014-01-01

    Background: Failure in the treatment of burn patients infected with Pseudomonas aeruginosa could happen as a result of the acquisition of antibiotic resistance, including carbapenems. Objectives: The aim of the present study was to investigate the phenotypic and genotypic characteristics of the Pseudomonas aeruginosa strains, isolated from burn patients. Patients and Methods: During a 12 month period, in this cross-sectional study, two hundred seventy strains of Pseudomonas aeruginosa were isolated from the burn patients in Ghotbeddin Burn Hospital, Shiraz, Iran. Screening for the carbapenem resistance in the isolates was carried out by the E test method. Sensitivity patterns of metallo-β-lactamase (MβLs) producing strains of pseudomonas to eleven antibiotics were determined by the mentioned method. The epidemiological associations of these strains were determined by Pulsed-field gel electrophoresis (PFGE). Results: Of the 270 strains, 60 (22.2%) were resistant to imipenem and meropenem, classified as MβLs producing. MβLs producing strains of pseudomonas were completely resistant to five tested antibiotics while their sensitivities to the three most effective antibiotics including ceftazidime, amikacin and ciprofloxacin were 23.4%, 6.7 % and 1.7%, respectively. In PFGE, 37 patterns from the genome of Pseudomonas aeruginosa were observed. Majority of the strains (43; 71.6%) exhibited more than 80% similarity, based on the drawn dendrogram. Conclusions: According to the results, none of the tested antibiotics is safe to prescribe. As PFGE revealed, a limited number of Pseudomonas aeruginosa types are predominant in the hospitals which infect the burn patients. PMID:25031843

  11. Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

    PubMed Central

    2011-01-01

    Background Our previously published reports have described an effective biocontrol agent named Pseudomonas sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of P. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures. Results The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved P. aeruginosa core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a capB gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other P. aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of P. aeruginosa strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C. Conclusions The P. aeruginosa strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche. PMID:21884571

  12. Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology

    PubMed Central

    Tayabali, Azam F.; Coleman, Gordon; Nguyen, Kathy C.

    2015-01-01

    Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications. PMID:26619347

  13. Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain

    PubMed Central

    Noguera, D. R.; Freedman, D. L.

    1996-01-01

    Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored. PMID:16535348

  14. Biodegradation and extracellular enzymatic activities of Pseudomonas aeruginosa strain GF31 on β-cypermethrin.

    PubMed

    Tang, Aixing; Wang, Bowen; Liu, Youyan; Li, Qingyun; Tong, Zhangfa; Wei, Yingjun

    2015-09-01

    Pseudomonas aeruginosa strain GF31, isolated from a contaminated soil, can effectively degrade β-cypermethrin (β-CP), as well as fenpropathrin, fenvalerate, and cyhalothrin. The highest level of degradation (81.2 %) was achieved with the addition of peptone. Surprisingly, the enzyme responsible for degradation was mainly localized to the extracellular areas of the bacteria, in contrast to the other known pyrethroid-degrading enzymes, which are intracellular. Although intact bacterial cells function at about 30 °C for biodegradation, similar to other degrading strains, the crude extracellular extract of strain GF31 remained biologically active at 60 °C. Moreover, the extract fraction showed good storage stability, maintaining >50 % of its initial activity following storage at 25 °C for at least 20 days. Significant differences in the characteristics of the crude GF31 extracellular extract compared with the known pyrethroid-degrading enzymes indicate the presence of a novel pyrethroid-degrading enzyme. Furthermore, the identification of 3-phenoxybenzoic acid and 2,2-dimethylcyclopropanecarboxylate from the degradation products suggests the possibility that β-CP degradation by both the strain and the crude extracellular fraction is achieved through a hydrolysis pathway. Further degradation of these two metabolites may lead to the development of an efficient method for the mineralization of these types of pollutants. PMID:25921758

  15. Draft Genome Sequence of Pseudomonas aeruginosa Strain N002, Isolated from Crude Oil-Contaminated Soil from Geleky, Assam, India

    PubMed Central

    Roy, Abhjit Sarma; Baruah, Reshita; Gogoi, Dhrubajyoti; Borah, Maina

    2013-01-01

    Here, we report the draft genome sequence of crude oil-degrading Pseudomonas aeruginosa strain N002, isolated from a crude oil-polluted soil sample from Geleky, Assam, India. Multiple genes potentially involved in crude oil degradation were identified. PMID:23405324

  16. Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa.

    PubMed

    Serino, L; Reimmann, C; Baur, H; Beyeler, M; Visca, P; Haas, D

    1995-11-15

    Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa. PMID:7500944

  17. Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

    PubMed

    Lødeng, A G G; Ahlén, C; Lysvand, H; Mandal, L H; Iversen, O J

    2006-08-01

    This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype. PMID:16842571

  18. The ferrichrome receptor A as a new target for Pseudomonas aeruginosa virulence attenuation.

    PubMed

    Lee, Keehoon; Lee, Kang-Mu; Go, Junhyeok; Ryu, Jae-Chan; Ryu, Ji-Hwan; Yoon, Sang Sun

    2016-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen. PMID:27190289

  19. Integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models reveals that pathogenicity is host specific.

    PubMed

    Dubern, Jean-Frédéric; Cigana, Cristina; De Simone, Maura; Lazenby, James; Juhas, Mario; Schwager, Stephan; Bianconi, Irene; Döring, Gerd; Eberl, Leo; Williams, Paul; Bragonzi, Alessandra; Cámara, Miguel

    2015-11-01

    Pseudomonas aeruginosa is a multi-host opportunistic pathogen causing a wide range of diseases because of the armoury of virulence factors it produces, and it is difficult to eradicate because of its intrinsic resistance to antibiotics. Using an integrated whole-genome approach, we searched for P. aeruginosa virulence genes with multi-host relevance. We constructed a random library of 57 360 Tn5 mutants in P. aeruginosa PAO1-L and screened it in vitro for those showing pleiotropic effects in virulence phenotypes (reduced swarming, exo-protease and pyocyanin production). A set of these pleiotropic mutants were assayed for reduced toxicity in Drosophila melanogaster, Caenorhabditis elegans, human cell lines and mice. Surprisingly, the screening revealed that the virulence of the majority of P. aeruginosa mutants varied between disease models, suggesting that virulence is dependent on the disease model used and hence the host environment. Genomic analysis revealed that these virulence-related genes encoded proteins from almost all functional classes, which were conserved among P. aeruginosa strains. Thus, we provide strong evidence that although P. aeruginosa is capable of infecting a wide range of hosts, many of its virulence determinants are host specific. These findings have important implication when searching for novel anti-virulence targets to develop new treatments against P. aeruginosa. PMID:25845292

  20. Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

    PubMed Central

    Lafleur, John; Lepidi, Hubert; Papazian, Laurent; Rolain, Jean-Marc; Raoult, Didier; Elias, Mikael; Silby, Mark W.; Bzdrenga, Janek; Bregeon, Fabienne; Chabriere, Eric

    2014-01-01

    Rationale The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia. Objectives The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia. Methods To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage. Measurements and Primary Results SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality. Conclusion These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use. PMID:25350373

  1. RESULTS OF MONITORING METALLO-BETA-LACTAMASE-PRODUCING STRAINS OF PSEUDOMONAS AERUGINOSA IN A MULTI-PROFILE HOSPITAL.

    PubMed

    Shamaeva, S K; Portnyagina, U S; Edelstein, M V; Kuzmina, A A; Maloguloval, S; Varfolomeeva, N A

    2015-01-01

    The authors present the results of long-term monitoring of metallo-beta-lactamase (MBL) producing strains of Pseudomonas aeruginosa in the Republican Hospital No 2 of Yakutsk, Russian Federation. Hospitals across Russia, as well as the rest of the world, face a rapid appearance and a virtually unchecked spread of multiresistant and panresistant nosocomial pathogens. Especially prevalent are multidrug-resistant isolates of P. aeruginosa, most often found among the patients of intensive care and intensive therapy units, as well as surgery departments. The aim of this study is to investigate the prevalence of metallo-beta-lactamase-producing strains of P. aeruginosa in a multi-profile hospital. 2,135 isolates of P. aeruginosa were studied, collected during a time span of seven years (2008-2014) from clinical specimens of hospitalised patients in acute surgery, purulent surgery, neurosurgery, otolaryngology, coloproctology departments, intensive care and intensive therapy, burn units, as well as intensive care unit for patients with acute cerebrovascular accidents and coronary care unit. Strains were identified and re-identified using established methods, NEFERMtest 24 (MICROLATEST) biochemical microtest and API (bioMerieux) test systems were used. For all carbapenem-resistant strains a phenotype screening for MBL was performed using the double-disks method with EDTA. In order to identify VIM-type and IMP-type MBL genes a real-time multiplex polymerase chain reaction was used. Among the investigated strains the largest number of P. aeruginosa - 35.6% (761 isolates) was found in patients at intensive care and intensive therapy units. Clonal expansion of extensively drug-resistant strain P. aeruginosa ST235 (VIM-2) was determined, the resistance mechanism of which is connected to MBL. Sensitivity determination of MBL-producing isolates of P. aeruginosa has shown that isolated strains have a high level of resistance (100%) to all tested antibacterial agents: piperacillin

  2. Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

    PubMed

    Okamoto, K; Gotoh, N; Tsujimoto, H; Yamada, H; Yoshihara, E; Nakae, T; Nishino, T

    1999-01-01

    We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics. PMID:10338201

  3. Production of biosurfactant from a new and promising strain of Pseudomonas aeruginosa PA1.

    PubMed

    Santa Anna, L M; Sebastian, G V; Pereira, N; Alves, T L; Menezes, E P; Freire, D M

    2001-01-01

    The Pseudomonas aeruginosa PA1 strain, isolated from the water of oil production in Sergipe, Northeast Brazil, was evaluated as a potential rhamnolipid type of biosurfactant producer. The production of biosurfactants was investigated using different carbon sources (n-hexadecane, paraffin oil, glycerol, and babassu oil) and inoculum concentrations (0.0016-0.008 g/L). The best results were obtained with glycerol as the substrate and an initial cell concentration of 0.004 g/L. A C:N ratio of 22.8 led to the greatest production of rhamnolipids (1700 mg/L) and efficiency (1.18 g of rhamnolipid/g of dry wt). PMID:11963874

  4. Assessment of the Effects of Light Availability on Growth and Competition Between Strains of Planktothrix agardhii and Microcystis aeruginosa.

    PubMed

    Torres, Camila de Araujo; Lürling, Miquel; Marinho, Marcelo Manzi

    2016-05-01

    In this study, we tested the hypothesis that Planktothrix agardhii strains isolated from a tropical water body were better competitors for light than Microcystis aeruginosa strains. These cyanobacteria are common in eutrophic systems, where light is one of the main drivers of phytoplankton, and Planktothrix is considered more shade-adapted and Microcystis more high-light tolerant. First, the effect of light intensities on growth was studied in batch cultures. Next, the minimum requirement of light (I*) and the effect of light limitation on the outcome of competition was investigated in chemostats. All strains showed similar growth at 10 μmol photons m(-2) s(-1), demonstrating the ability of the two species to grow in low light. The optimum light intensity was lower for P. agardhii, but at the highest light intensity, Microcystis strains reached higher biovolume, confirming that P. agardhii has higher sensitivity to high light. Nonetheless, P. agardhii grew in light intensities considered high (500 μmol photons m(-2) s(-1)) for this species. M. aeruginosa showed a higher carrying capacity in light-limited condition, but I* was similar between all the strains. Under light competition, Microcystis strains displaced P. agardhii and dominated. In two cases, there was competitive exclusion and in the other two P. agardhii managed to remain in the system with a low biovolume (≈15 %). Our findings not only show that strains of P. agardhii can grow under higher light intensities than generally assumed but also that strains of M. aeruginosa are better competitors for light than supposed. These results help to understand the co-occurrence of these species in tropical environments and the dominance of M. aeruginosa even in low-light conditions. PMID:26691315

  5. Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa

    PubMed Central

    Dehner, Carolyn; Morales-Soto, Nydia; Behera, Rabindra K.; Shrout, Joshua; Theil, Elizabeth C.; Maurice, Patricia A.

    2013-01-01

    Metabolism of iron derived from insoluble and/ or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O2 or H2O2. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high. PMID:23417538

  6. The D3 bacteriophage α-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa.

    PubMed

    Taylor, Véronique L; Udaskin, Molly L; Islam, Salim T; Lam, Joseph S

    2013-10-01

    Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector. Increased induction of Iap expression completely abrogated OSA production in the wzz1 wzz2 double mutant, while background levels of OSA production were still observed in either of the single mutants. Therefore, Iap inhibition of OSA biosynthesis was most effective in the absence of both Wzz proteins. Sequence alignment analyses revealed a high degree of similarity between Iap and the first transmembrane segment (TMS) of either Wzz1 or Wzz2. Various topology prediction analyses of the Iap sequence consistently predicted the presence of a single TMS, suggesting a propensity for Iap to insert itself into the inner membrane (IM). The compromised ability of Iap to abrogate Wzyα function in the presence of Wzz1 or Wzz2 provides compelling evidence that inhibition occurs after Wzyα inserts itself into the IM and is achieved through mimicry of the first TMS from the Wzz proteins of P. aeruginosa PAO1. PMID:23955007

  7. Structure and fate of a Pseudomonas aeruginosa population originating from a combined sewer and colonizing a wastewater treatment lagoon.

    PubMed

    Lavenir, Raphaël; Petit, Stéphanie M-C; Alliot, Nolwenn; Ribun, Sébastien; Loiseau, Laurence; Marjolet, Laurence; Briolay, Jérôme; Nazaret, Sylvie; Cournoyer, Benoit

    2014-04-01

    The efficacy of a wastewater treatment lagoon (WWTL) at preventing the spread of Pseudomonas aeruginosa into natural aquatic habitats was investigated. A WWTL and its connected combined sewer and brook were exhaustively sampled. Physico-chemical analyses showed a stratification of the first pond according to pH, temperature and oxygen content. The P. aeruginosa counts partially matched this stratification with higher values among the bottom anaerobic waters of the first half of this pond. Genotyping of 494 WWTL P. aeruginosa strains was performed and led to the definition of 85 lineages. Dominant lineages were observed, with some being found all over the WWTL including the connected brook. IS5 was used as an indicator of genomic changes, and 1 to 12 elements were detected among 16 % of the strains. IS-driven lasR (genetic regulator) disruptions were detected among nine strains that were not part of the dominant lineages. These insertional mutants did not show significant elastase activities but showed better growth than the PAO1 reference strain in WWTL waters. Differences in growth patterns were related to a better survival of these mutants at an alkaline pH and a better ability at using some C-sources such as alanine. The opportunistic colonization of a WWTL by P. aeruginosa can involve several metabolic strategies which appeared lineage specific. Some clones appeared more successful than others at disseminating from a combined sewer toward the overflow of a WWTL. PMID:24407782

  8. Serotyping of Pseudomonas aeruginosa strains isolated in Bulgaria using the Lányi-Bergan combined scheme.

    PubMed

    Pencheva, P

    1986-01-01

    Two hundred Pseudomonas aeruginosa strains isolated in hospitals in Bulgaria were serotyped according to the combined scheme of Lányi and Bergan, supplemented by Akatova and Smirnova and Homma, using agglutinating O-antisera prepared in the National Institute of Hygiene, Budapest. The most frequently encountered serogroup is O2 (29%) followed by O11 (28.5%), O6, O3, O10 etc. The results were compared with those obtained by using Difco antisera prepared according to Liu et al., and showed 96.5% coincidence. The strains were phage typed according to the scheme of Meitert and tested for antibiotic resistance to aminoglycosides (gentamicin, carbenicillin, tobramycin and amikacin). Phage groups 3 (3a and 3(3)) and 1 (1a) predominated. The strains exhibited sensitivity to amikacin (99%) and frequent resistance to gentamicin (45.8%, carbenicillin (40%) and tobramycin (28%). Subdivision of the serogroups into phage and resisto-types contributes to analysis of nosocomial infections. PMID:3115050

  9. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence.

    PubMed

    Lee, Jintae; Attila, Can; Cirillo, Suat L G; Cirillo, Jeffrey D; Wood, Thomas K

    2009-01-01

    Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  10. Engineered biosealant strains producing inorganic and organic biopolymers.

    PubMed

    Bergdale, Terran E; Pinkelman, Rebecca J; Hughes, Stephen R; Zambelli, Barbara; Ciurli, Stefano; Bang, Sookie S

    2012-10-31

    Microbiologically induced calcium carbonate precipitation (MICCP) is a naturally occurring biological process that has shown its potential in remediation of a wide range of structural damages including concrete cracks. In this study, genetically engineered microorganisms, capable of producing extracellular polymeric substances (EPSs) as well as inducing MICCP, were developed based on the assumption that the complex of inorganic CaCO(3) and organic EPS would provide a stronger matrix than MICCP alone as biosealant. In order to develop a recombinant biosealant microorganism, the entire Sporosarcina pasteurii urease gene sequences including ureA, ureB, ureC, ureD, ureE, ureF, and ureG from plasmid pBU11 were sub-cloned into the shuttle vector, pUCP18. The newly constructed plasmid, pUBU1, was transformed into two Pseudomonas aeruginosa strains, 8821 and PAO1, to develop recombinants capable of inducing calcite precipitation in addition to their own ability to produce EPS. Nickel-dependent urease activities were expressed from the recombinant P. aeruginosa 8821 (pUBU1) and P. aeruginosa PAO1 (pUBU1), at 99.4% and 60.9% of the S. pasteurii urease activity, respectively, in a medium containing 2mM NiCl(2). No urease activities were detected from the wild type P. aeruginosa 8821 and P. aeruginosa PAO1 under the same growth conditions. Recombinant Pseudomonas strains induced CaCO(3) precipitation at a comparable rate as S. pasteurii and scanning electron microscopy evidenced the complex of CaCO(3) crystals and EPS layers surrounding the cells. The engineered strains produced in this study are expected to serve as a valuable reference to future biosealants that could be applied in the environment. However, the pathogenic potential of P. aeruginosa, used here only as a model system to show the proof of principle, prevents the use of this recombinant organism as a biosealant. In practical applications, other recombinant organisms should be used. PMID:22789480

  11. Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases

    PubMed Central

    Huang, Cheng-Yang

    2015-01-01

    Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the Kd value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases. PMID:25993634

  12. Effects of Quorum Sensing Systems on Regulatory T Cells in Catheter-Related Pseudomonas aeruginosa Biofilm Infection Rat Models

    PubMed Central

    Feng, Lei; Xiang, Qingqing; Ai, Qing; Wang, Zhengli; Zhang, Yunhui; Lu, Qi

    2016-01-01

    Background. Quorum sensing (QS) systems play an important role in modulating biofilm formation. Recent studies have found that the QS molecules had complex effects on the host immune systems. In addition, regulatory T cells (Tregs), known as important negative regulators in the immune system, have been found upregulated in multiple chronic infections. Therefore, the QS systems were hypothesized to be involved in modulating Tregs in biofilm-associated infections. Object. To explore the effects of QS systems on Tregs in catheter-related Pseudomonas aeruginosa biofilm infection rat models. Method. Catheter-related Pseudomonas aeruginosa biofilm infection rat models were established; the bacterial clearance rates, total cell counts in bronchoalveolar lavage (BAL) fluid, pathological changes of lungs, and the levels of Foxp3, TGF-β1, and IL-10 in PAO1 strain group were examined and compared with the QS-mutant ΔlasRΔrhlR and ΔlasIΔrhlI groups. Results. In PAO1 group, the bacterial clearance rates were lower, total cell counts were higher, pathological changes were severer, and the levels of Foxp3, TGF-β1, and IL-10 were significantly higher compared with QS-mutant groups (p < 0.05). No significant difference was observed between the two QS-mutant groups (p > 0.05). Conclusion. QS systems can trigger host immune system, accompanied with the upregulation of Tregs. PMID:27069314

  13. Genome Sequence of a Virulent Pseudomonas aeruginosa Strain, 12-4-4(59), Isolated from the Blood Culture of a Burn Patient

    PubMed Central

    Karna, S. L. Rajasekhar; Chen, Tsute; Chen, Ping; Peacock, Trent J.; Abercrombie, Johnathan J.

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that frequently infects wounds, significantly impairs wound healing, and causes morbidity and mortality in burn patients. Here, we report the genome sequence of a virulent strain of P. aeruginosa, 12-4-4(59), isolated from the blood culture of a burn patient. PMID:26941150

  14. Absence of Membrane Phosphatidylcholine Does Not Affect Virulence and Stress Tolerance Phenotypes in the Opportunistic Pathogen Pseudomonas aeruginosa

    PubMed Central

    Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.

    2012-01-01

    During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline. PMID:22363496

  15. Screening of Lactobacillus spp. for the prevention of Pseudomonas aeruginosa pulmonary infections

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host’s health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. Results Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. Conclusion Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further

  16. A novel protein quality control mechanism contributes to heat shock resistance of worldwide-distributed Pseudomonas aeruginosa clone C strains.

    PubMed

    Lee, Changhan; Wigren, Edvard; Trček, Janja; Peters, Verena; Kim, Jihong; Hasni, Muhammad Sharif; Nimtz, Manfred; Lindqvist, Ylva; Park, Chankyu; Curth, Ute; Lünsdorf, Heinrich; Römling, Ute

    2015-11-01

    Pseudomonas aeruginosa is a highly successful nosocomial pathogen capable of causing a wide variety of infections with clone C strains most prevalent worldwide. In this study, we initially characterize a molecular mechanism of survival unique to clone C strains. We identified a P. aeruginosa clone C-specific genomic island (PACGI-1) that contains the highly expressed small heat shock protein sHsp20c, the founding member of a novel subclass of class B bacterial small heat shock proteins. sHsp20c and adjacent gene products are involved in resistance against heat shock. Heat stable sHsp20c is unconventionally expressed in stationary phase in a wide temperature range from 20 to 42°C. Purified sHsp20c has characteristic features of small heat shock protein class B as it is monodisperse, forms sphere-like 24-meric oligomers and exhibits significant chaperone activity. As the P. aeruginosa clone C population is significantly more heat shock resistant than genetically unrelated P. aeruginosa strains without sHsp20c, the horizontally acquired shsp20c operon might contribute to the survival of worldwide-distributed clone C strains. PMID:26014207

  17. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

    PubMed

    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p<0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections. PMID:25704369

  18. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa. PMID:25573142

  19. Detection of Quorum Sensing Activity in the Multidrug-Resistant Clinical Isolate Pseudomonas aeruginosa Strain GB11

    PubMed Central

    Cheng, Huey Jia; Ee, Robson; Cheong, Yuet Meng; Tan, Wen-Si; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    A multidrug-resistant clinical bacteria strain GB11 was isolated from a wound swab on the leg of a patient. Identity of stain GB11 as Pseudomonas aeruginosa was validated by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Detection of the production of signaling molecules, N-acylhomoserine lactones (AHLs), was conducted using three different bacterial biosensors. A total of four different AHLs were found to be produced by strain GB11, namely N-butyryl homoserine lactone (C4-HSL), N-hexanoylhomoserine lactone (C6-HSL), N-octanoyl homoserine lactone (C8-HSL) and N-3-oxo-dodecanoylhomoserine lactone (3-oxo-C12-HSL) using high resolution liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these detected AHLs, 3-oxo-C12-HSL was found to be the most abundant AHL produced by P. aeruginosa GB11. PMID:25019635

  20. Pseudomonas aeruginosa Cytotoxicity Is Attenuated at High Cell Density and Associated with the Accumulation of Phenylacetic Acid

    PubMed Central

    Wang, Jianhe; Dong, Yihu; Zhou, Tielin; Liu, Xiaoling; Deng, Yinyue; Wang, Chao; Lee, Jasmine; Zhang, Lian-Hui

    2013-01-01

    Background P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. Methodology/Findings Intriguingly, however, in this study we noticed that while a low cell density inoculum of P. aeruginosa caused severe cytotoxicity against human lung tissue cell line A549, increasing the cell density of bacterial inoculum led to decreased cytotoxicity. Addition of the supernatants from high density bacterial culture to low cell density inoculum protected the human cells from bacterial cytotoxic damage, suggesting that P. aeruginosa may produce and accumulate an inhibitory molecule(s) counteracting its pathogenic infection. The inhibitor was purified from the stationary-phase culture supernatants of P. aeruginosa strain PAO1 using bioassay-guided high performance liquid chromatography (HPLC), and characterized to be phenylacetic acid (PAA) by mass spectrometry and nuclear magnetic resonance spectroscopy. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of Type III secretion system (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Conclusions Identification of bacterial metabolite PAA as a T3SS-specific inhibitor explains this intriguing inverse cell-density-dependent-cytotoxicity phenomenon as T3SS is known to be a key virulence factor associated with cytotoxicity and acute infection. The findings may provide useful clues for design and development of new strategies to combat this formidable bacterial pathogen. PMID:23555919

  1. Environmentally Endemic Pseudomonas aeruginosa Strains with Mutations in lasR Are Associated with Increased Disease Severity in Corneal Ulcers

    PubMed Central

    Hammond, John H.; Hebert, Wesley P.; Naimie, Amanda; Ray, Kathryn; Van Gelder, Rachel D.; DiGiandomenico, Antonio; Lalitha, Prajna; Srinivasan, Muthiah; Acharya, Nisha R.; Lietman, Thomas; Hogan, Deborah A.

    2016-01-01

    ABSTRACT The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from

  2. Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

    PubMed Central

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  3. Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

    PubMed

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  4. Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.

    PubMed

    Kolpen, Mette; Mousavi, Nabi; Sams, Thomas; Bjarnsholt, Thomas; Ciofu, Oana; Moser, Claus; Kühl, Michael; Høiby, Niels; Jensen, Peter Østrup

    2016-02-01

    Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections. PMID:26774522

  5. N2-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa

    PubMed Central

    Jann, Alfred; Stalon, Victor; Wauven, Corinne Vander; Leisinger, Thomas; Haas, Dieter

    1986-01-01

    Arginine-nonutilizing (aru) mutants of Pseudomonas aeruginosa strain PAO converted L-arginine to N2-succinylarginine or N-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of N2-succinylornithine. Enzyme assays with crude P. aeruginosa extracts established the following pathway: L-arginine + succinyl-CoA → N2-succinylarginine → N2-succinylornithine → N_succinylglutamate 5-semialdehyde → N-succinylglutamate → succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified N2-acetylornithine 5-aminotransferase (N2-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated N2-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables P. aeruginosa to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in P. aeruginosa fulfill different functions. Images PMID:16593724

  6. Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

    PubMed

    Lassek, Christian; Berger, Antje; Zühlke, Daniela; Wittmann, Christoph; Riedel, Katharina

    2016-05-01

    Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 (http://proteomecentral.proteomexchange.org/dataset/PXD002373). PMID:26959854

  7. Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

    PubMed

    Heurlier, Karin; Dénervaud, Valérie; Haenni, Marisa; Guy, Lionel; Krishnapillai, Viji; Haas, Dieter

    2005-07-01

    In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants. PMID:15995202

  8. Silver Nanoparticles: Biosynthesis Using an ATCC Reference Strain of Pseudomonas aeruginosa and Activity as Broad Spectrum Clinical Antibacterial Agents

    PubMed Central

    Quinteros, Melisa A.; Aiassa Martínez, Ivana M.; Dalmasso, Pablo R.; Páez, Paulina L.

    2016-01-01

    Currently, the biosynthesis of silver-based nanomaterials attracts enormous attention owing to the documented antimicrobial properties of these ones. This study reports the extracellular biosynthesis of silver nanoparticles (Ag-NPs) using a Pseudomonas aeruginosa strain from a reference culture collection. A greenish culture supernatant of P. aeruginosa incubated at 37°C with a silver nitrate solution for 24 h changed to a yellowish brown color, indicating the formation of Ag-NPs, which was confirmed by UV-vis spectroscopy, transmission electron microscopy, and X-ray diffraction. TEM analysis showed spherical and pseudospherical nanoparticles with a distributed size mainly between 25 and 45 nm, and the XRD pattern revealed the crystalline nature of Ag-NPs. Also it provides an evaluation of the antimicrobial activity of the biosynthesized Ag-NPs against human pathogenic and opportunistic microorganisms, namely, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Proteus mirabilis, Acinetobacter baumannii, Escherichia coli, P. aeruginosa, and Klebsiella pneumonia. Ag-NPs were found to be bioactive at picomolar concentration levels showing bactericidal effects against both Gram-positive and Gram-negative bacterial strains. This work demonstrates the first helpful use of biosynthesized Ag-NPs as broad spectrum bactericidal agents for clinical strains of pathogenic multidrug-resistant bacteria such as methicillin-resistant S. aureus, A. baumannii, and E. coli. In addition, these Ag-NPs showed negligible cytotoxic effect in human neutrophils suggesting low toxicity to the host. PMID:27340405

  9. Silver Nanoparticles: Biosynthesis Using an ATCC Reference Strain of Pseudomonas aeruginosa and Activity as Broad Spectrum Clinical Antibacterial Agents.

    PubMed

    Quinteros, Melisa A; Aiassa Martínez, Ivana M; Dalmasso, Pablo R; Páez, Paulina L

    2016-01-01

    Currently, the biosynthesis of silver-based nanomaterials attracts enormous attention owing to the documented antimicrobial properties of these ones. This study reports the extracellular biosynthesis of silver nanoparticles (Ag-NPs) using a Pseudomonas aeruginosa strain from a reference culture collection. A greenish culture supernatant of P. aeruginosa incubated at 37°C with a silver nitrate solution for 24 h changed to a yellowish brown color, indicating the formation of Ag-NPs, which was confirmed by UV-vis spectroscopy, transmission electron microscopy, and X-ray diffraction. TEM analysis showed spherical and pseudospherical nanoparticles with a distributed size mainly between 25 and 45 nm, and the XRD pattern revealed the crystalline nature of Ag-NPs. Also it provides an evaluation of the antimicrobial activity of the biosynthesized Ag-NPs against human pathogenic and opportunistic microorganisms, namely, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Proteus mirabilis, Acinetobacter baumannii, Escherichia coli, P. aeruginosa, and Klebsiella pneumonia. Ag-NPs were found to be bioactive at picomolar concentration levels showing bactericidal effects against both Gram-positive and Gram-negative bacterial strains. This work demonstrates the first helpful use of biosynthesized Ag-NPs as broad spectrum bactericidal agents for clinical strains of pathogenic multidrug-resistant bacteria such as methicillin-resistant S. aureus, A. baumannii, and E. coli. In addition, these Ag-NPs showed negligible cytotoxic effect in human neutrophils suggesting low toxicity to the host. PMID:27340405

  10. Enhancement of Rhamnolipid Production in Residual Soybean Oil by an Isolated Strain of Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    de Lima, C. J. B.; França, F. P.; Sérvulo, E. F. C.; Resende, M. M.; Cardoso, V. L.

    In the present work, the production of rhamnolipid from residual soybean oil (RSO) from food frying facilities was studied using a strain of Pseudomonas aeruginosa of contaminated lagoon, isolated from a hydrocarbon contaminated soil. The optimization of RSO, amonium nitrate, and brewery residual yeast concentrations was accomplished by a central composite experimental design and surface response analysis. The experiments were performed in 500-mL Erlenmeyer flasks containing 50mL of mineral medium, at 170 rpm and 30±1°C, for a 48-h fermentation period. Rhamnolipid production has been monitored by measurements of surface tension, rhamnose concentration, and emulsifying activity. The best-planned results, located on the central point, have corresponded to 22g/L of RSO, 5.625 g/ L of NH4NO3' and 11.5 g/L of brewery yeast. At the maximum point the values for rhamnose and emulsifying index were 2.2g/L and 100%, respectively.

  11. Sodium Nitrite-Mediated Killing of the Major Cystic Fibrosis Pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under Anaerobic Planktonic and Biofilm Conditions▿

    PubMed Central

    Major, Tiffany A.; Panmanee, Warunya; Mortensen, Joel E.; Gray, Larry D.; Hoglen, Niel; Hassett, Daniel J.

    2010-01-01

    A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO2−) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO2− (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 μg ml−1 NO2−). B. cepacia was particularly sensitive to low concentrations of A-NO2− (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO2− killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO2−. Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO2− can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO2− is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO2−, nitric oxide (NO), are dramatically reduced. PMID:20696868

  12. Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa.

    PubMed

    Boukerb, Amine M; Decor, Aude; Ribun, Sébastien; Tabaroni, Rachel; Rousset, Audric; Commin, Loris; Buff, Samuel; Doléans-Jordheim, Anne; Vidal, Sébastien; Varrot, Annabelle; Imberty, Anne; Cournoyer, Benoit

    2016-01-01

    LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewis(a) rather than Lewis(x). Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewis(a) showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great

  13. Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa

    PubMed Central

    Boukerb, Amine M.; Decor, Aude; Ribun, Sébastien; Tabaroni, Rachel; Rousset, Audric; Commin, Loris; Buff, Samuel; Doléans-Jordheim, Anne; Vidal, Sébastien; Varrot, Annabelle; Imberty, Anne; Cournoyer, Benoit

    2016-01-01

    LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewisa rather than Lewisx. Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewisa showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations

  14. A Pseudomonas aeruginosa EF-Hand Protein, EfhP (PA4107), Modulates Stress Responses and Virulence at High Calcium Concentration

    PubMed Central

    Sarkisova, Svetlana A.; Lotlikar, Shalaka R.; Guragain, Manita; Kubat, Ryan; Cloud, John

    2014-01-01

    Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+]. PMID

  15. Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats.

    PubMed Central

    Kelly, N M; Bell, A; Hancock, R E

    1989-01-01

    Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum. Images PMID:2492257

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

    PubMed

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

    2015-01-01

    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. PMID:25464282

  17. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

    PubMed Central

    Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza; Horatzek, Sonja; Salunkhe, Prabhakar; Munder, Antje; van Barneveld, Andrea; Jordan, Doris; Bredenbruch, Florian; Häußler, Susanne; Riedel, Kathrin; Eberl, Leo; Jensen, Peter Østrup; Bjarnsholt, Thomas; Moser, Claus; Hoiby, Niels; Tümmler, Burkhard; Wiehlmann, Lutz

    2008-01-01

    Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population. PMID:19054330

  18. The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection.

    PubMed

    Rodríguez-Rojas, Alexandro; Blázquez, Jesús

    2009-02-01

    Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation. PMID:19028889

  19. Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung

    PubMed Central

    Dingemans, Jozef; Monsieurs, Pieter; Yu, Sung-Huan; Crabbé, Aurélie; Förstner, Konrad U.; Malfroot, Anne

    2016-01-01

    ABSTRACT Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. PMID:27486191

  20. Impact of higher alginate expression on deposition of Pseudomonas aeruginosa in radial stagnation point flow and reverse osmosis systems.

    PubMed

    Herzberg, Moshe; Rezene, Tesfalem Zere; Ziemba, Christopher; Gillor, Osnat; Mathee, Kalai

    2009-10-01

    Extracellular polymeric substances (EPS) have major impact on biofouling of reverse osmosis (RO) membranes. On one hand, EPS can reduce membrane permeability and on the other, EPS production by the primary colonizers may influence their deposition and attachment rate and subsequently affect the biofouling propensity of the membrane. The role of bacterial exopolysaccharides in bacterial deposition followed by the biofouling potential of an RO membrane was evaluated using an alginate overproducing (mucoid) Pseudomonas aeruginosa. The mucoid P. aeruginosa PAOmucA22 was compared with its isogenic nonmucoid prototypic parent PAO1 microscopically in a radial stagnation point flow (RSPF) system for their bacterial deposition characteristics. Then, biofouling potential of PAO1 and PAOmucA22 was determined in a crossflow rectangular plate-and-frame membrane cell, in which the strains were cultivated on a thin-film composite, polyamide, flat RO membrane coupon (LFC-1) under laminar flow conditions. In the RSPF system, the observed deposition rate of the mucoid strain was between 5- and 10-fold lower than of the wild type using either synthetic wastewater medium (with ionic strength of 14.7 mM and pH 7.4) or 15 mM KCl solution (pH of 6.2). The slower deposition rate of the mucoid strain is explained by 5- to 25-fold increased hydrophilicity of the mucoid strain as compared to the isogenic wild type, PAO1. Corroborating with these results, a significant delay in the onset of biofouling of the RO membrane was observed when the mucoid strain was used as the membrane colonizer, in which the observed time for the induced permeate flux decline was delayed (ca. 2-fold). In conclusion, the lower initial cell attachment of the mucoid strain decelerated biofouling of the RO membrane. Bacterial deposition and attachment is a critical step in biofilm formation and governed by intimate interactions between outer membrane proteins of the bacteria and the surface. Shielding these

  1. The MSHA strain of Pseudomonas aeruginosa activated TLR pathway and enhanced HIV-1 DNA vaccine immunoreactivity.

    PubMed

    Hou, Jue; Liu, Yong; Liu, Ying; Shao, Yiming

    2012-01-01

    The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells) and antigen presenting cells (APCs). Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR) pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs) following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells. PMID:23077664

  2. Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

    PubMed

    Elfarash, Ameer; Dingemans, Jozef; Ye, Lumeng; Hassan, Ahmed Amir; Craggs, Michael; Reimmann, Cornelia; Thomas, Mark S; Cornelis, Pierre

    2014-02-01

    Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain. PMID:24217175

  3. Biodegradation of isoproturon using a novel Pseudomonas aeruginosa strain JS-11 as a multi-functional bioinoculant of environmental significance.

    PubMed

    Dwivedi, Sourabh; Singh, Braj Raj; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2011-01-30

    Biodegradation of phenylurea herbicide isoproturon was studied in soil microcosm bioaugmented with a novel bacterial strain JS-11 isolated from wheat rhizosphere. The molecular characterization based on 16SrDNA sequence homology confirmed its identity as Pseudomonas aeruginosa strain JS-11. The herbicide was completely degraded within 20 days at ambient temperature with the rate constant of 0.08 day(-1), following the first-order rate kinetics. In stationary phase, at a cell density of 6.5 × 10(9) CFU mL(-1), the bacteria produced substantially increased amounts of indole acetic acid (IAA) in the presence of tryptophan as compared with the control. Also, the bacteria exhibited a time-dependent increase in the amount of tri-calcium phosphate solubilization in Pikovskaya's medium. Further screening of the strain JS-11 for auxiliary activities revealed its remarkable capability of producing the siderophores and hydrogen cyanide (HCN), besides antifungal activity against a common phytopathogen Fusarium oxysporum. Thus, the versatile P. aeruginosa strain JS-11 with innate potential for multifarious biological activities is envisaged as a super-bioinoculant for exploitation in the integrated bioremediation, plant growth and disease management (IBPDM) in contaminated agricultural soils. PMID:21035259

  4. Twenty-Five-Year Outbreak of Pseudomonas aeruginosa Infecting Individuals with Cystic Fibrosis: Identification of the Prairie Epidemic Strain

    PubMed Central

    Glezerson, Bryan A.; Sibley, Christopher D.; Sibley, Kristen A.; Duong, Jessica; Purighalla, Swathi; Mody, Christopher H.; Workentine, Matthew L.; Storey, Douglas G.; Surette, Michael G.; Rabin, Harvey R.

    2014-01-01

    Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection. PMID:24452167

  5. Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa

    PubMed Central

    Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

    2015-01-01

    Background: The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. Objectives: In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. Materials and Methods: A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. Results: The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Conclusions: Our findings indicate that probiotic bacterial

  6. X-ray Irradiated Vaccine Confers protection against Pneumonia caused by Pseudomonas Aeruginosa

    PubMed Central

    Li, Yanyan; Wang, Zhenling; Liu, Xiaoxiao; Tang, Jianying; Peng, Bin; Wei, Yuquan

    2016-01-01

    Pseudomonas aeruginosa is a gram-negative bacterium and one of the leading causes of nosocomial infection worldwide, however, no effective vaccine is currently available in the market. Here, we demonstrate that inactivation of the bacteria by X-ray irradiation inhibits its replication capability but retained antigenic expression functionally thus allowing its use as a potential vaccine. Mice immunized by this vaccine were challenged by the parental strain, the O-antigen-homologous strain PAO-1 (O2/O5) and heterologous strain PAO-6 (O6) in an acute pneumonia model. We further measured the protective effect of the vaccine, as well as host innate and cellular immunity responses. We found immunized mice could protect against both strains. Notably, the antiserum only had significant protective role against similar bacteria, while adoptive transfer of lymphocytes significantly controlled the spread of the virulent heterologous serogroup PAO-6 infection, and the protective role could be reversed by CD4 rather than CD8 antibody. We further revealed that vaccinated mice could rapidly recruit neutrophils to the airways early after intranasal challenge by PAO-6, and the irradiated vaccine was proved to be protective by the generated CD4+ IL-17+ Th17 cells. In conclusion, the generation of inactivated but metabolically active microbes is a promising strategy for safely vaccinating against Pseudomonas aeruginosa. PMID:26879055

  7. Whole-Genome Sequence of Multidrug-Resistant Pseudomonas aeruginosa Strain BAMCPA07-48, Isolated from a Combat Injury Wound

    PubMed Central

    Sanjar, Fatemeh; Karna, S. L. Rajasekhar; Chen, Tsute; Chen, Ping; Abercrombie, Johnathan J.

    2016-01-01

    We report here the complete genome sequence of Pseudomonas aeruginosa strain BAMCPA07-48, isolated from a combat injury wound. The closed genome sequence of this isolate is a valuable resource for pathogenome characterization of P. aeruginosa associated with wounds, which will aid in the development of a higher-resolution phylogenomic framework for molecular-guided pathogen-surveillance. PMID:27389262

  8. Rapid detection of Pseudomonas aeruginosa biomarkers in biological fluids using surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Wu, Xiaomeng; Chen, Jing; Zhao, Yiping; Zughaier, Susu M.

    2014-05-01

    Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes major infection not only in Cystic Fibrosis patients but also in chronic obstructive pulmonary disease and in critically ill patients in intensive care units. Successful antibiotic treatment of the infection relies on accurate and rapid identification of the infectious agents. Conventional microbiological detection methods usually take more than 3 days to obtain accurate results. We have developed a rapid diagnostic technique based on surface-enhanced Raman scattering to directly identify PA from biological fluids. P. aeruginosa strains, PAO1 and PA14, are cultured in lysogeny broth, and the SERS spectra of the broth show the signature Raman peaks from pyocyanin and pyoverdine, two major biomarkers that P. aeruginosa secretes during its growth, as well as lipopolysaccharides. This provides the evidence that the presence of these biomarkers can be used to indicate P. aeruginosa infection. A total of 22 clinical exhaled breath condensates (EBC) samples were obtained from subjects with CF disease and from non-CF healthy donors. SERS spectra of these EBC samples were obtained and further analyzed by both principle component analysis and partial least square-discriminant analysis (PLS-DA). PLS-DA can discriminate the samples with P. aeruginosa infection and the ones without P. aeruginosa infection at 99.3% sensitivity and 99.6% specificity. In addition, this technique can also discriminate samples from subject with CF disease and healthy donor with 97.5% sensitivity and 100% specificity. These results demonstrate the potential of using SERS of EBC samples as a rapid diagnostic tool to detect PA infection.

  9. The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

    PubMed Central

    Darzins, A

    1993-01-01

    The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili. Images PMID:8104179

  10. Nanoscale analysis of the effects of antibiotics and CX1 on a Pseudomonas aeruginosa multidrug-resistant strain

    NASA Astrophysics Data System (ADS)

    Formosa, C.; Grare, M.; Jauvert, E.; Coutable, A.; Regnouf-de-Vains, J. B.; Mourer, M.; Duval, R. E.; Dague, E.

    2012-08-01

    Drug resistance is a challenge that can be addressed using nanotechnology. We focused on the resistance of the bacteria Pseudomonas aeruginosa and investigated, using Atomic Force Microscopy (AFM), the behavior of a reference strain and of a multidrug resistant clinical strain, submitted to two antibiotics and to an innovative antibacterial drug (CX1). We measured the morphology, surface roughness and elasticity of the bacteria under physiological conditions and exposed to the antibacterial molecules. To go further in the molecules action mechanism, we explored the bacterial cell wall nanoscale organization using functionalized AFM tips. We have demonstrated that affected cells have a molecularly disorganized cell wall; surprisingly long molecules being pulled off from the cell wall by a lectin probe. Finally, we have elucidated the mechanism of action of CX1: it destroys the outer membrane of the bacteria as demonstrated by the results on artificial phospholipidic membranes and on the resistant strain.

  11. The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

    PubMed Central

    Sullivan, Aaron B.; Tam, K. P. Connie; Metruccio, Matteo M. E.; Evans, David J.

    2015-01-01

    Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88−/− epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88−/− mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain. PMID:25667266

  12. Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis.

    PubMed

    Wright, Elli A; Di Lorenzo, Valeria; Trappetti, Claudia; Liciardi, Manuele; Orru, Germano; Viti, Carlo; Bronowski, Christina; Hall, Amanda J; Darby, Alistair C; Oggioni, Marco R; Winstanley, Craig

    2015-01-30

    Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections. PMID:25475851

  13. The synergistic effect of visible light and gentamycin on Pseudomona aeruginosa microorganisms.

    PubMed

    Reznick, Yana; Banin, Ehud; Lipovsky, Anat; Lubart, Rachel; Polak, Pazit; Zalevsky, Zeev

    2013-01-01

    Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens(1-5). The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region(4,6,7). There are also reports of biocidal effect of red and near infra red(8) as well as green light(9). In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems(10). The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's. The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light. PMID:23852319

  14. The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

    PubMed Central

    Reznick, Yana; Banin, Ehud; Lipovsky, Anat; Lubart, Rachel; Polak, Pazit; Zalevsky, Zeev

    2013-01-01

    Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9. In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10. The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's. The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light. PMID:23852319

  15. Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon.

    PubMed

    Xu, Binjie; Wozniak, Daniel J

    2015-01-01

    Twitching motility is an important migration mechanism for the Gram-negative bacterium Pseudomonas aeruginosa. In the commonly used subsurface twitching assay, the sub-population of P. aeruginosa with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple P. aeruginosa virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was lecB, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates lecB transcription by directly binding to its promoter. The lecB gene was previously shown to be required for twitching motility in P. aeruginosa strain PAK; however, our lecB deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined. PMID:26309248

  16. Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon

    PubMed Central

    Xu, Binjie; Wozniak, Daniel J.

    2015-01-01

    Twitching motility is an important migration mechanism for the Gram-negative bacterium Pseudomonas aeruginosa. In the commonly used subsurface twitching assay, the sub-population of P. aeruginosa with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple P. aeruginosa virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was lecB, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates lecB transcription by directly binding to its promoter. The lecB gene was previously shown to be required for twitching motility in P. aeruginosa strain PAK; however, our lecB deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined. PMID:26309248

  17. Preparation, characterization and in vitro antimicrobial activity of liposomal ceftazidime and cefepime against Pseudomonas aeruginosa strains

    PubMed Central

    Torres, Ieda Maria Sapateiro; Bento, Etiene Barbosa; Almeida, Larissa da Cunha; de Sá, Luisa Zaiden Carvalho Martins; Lima, Eliana Martins

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic microorganism with the ability to respond to a wide variety of environmental changes, exhibiting a high intrinsic resistance to a number of antimicrobial agents. This low susceptibility to antimicrobial substances is primarily due to the low permeability of its outer membrane, efflux mechanisms and the synthesis of enzymes that promote the degradation of these drugs. Cephalosporins, particularty ceftazidime and cefepime are effective against P. aeruginosa, however, its increasing resistance has limited the usage of these antibiotics. Encapsulating antimicrobial drugs into unilamellar liposomes is an approach that has been investigated in order to overcome microorganism resistance. In this study, antimicrobial activity of liposomal ceftazidime and cefepime against P. aeruginosa ATCC 27853 and P. aeruginosa SPM-1 was compared to that of the free drugs. Liposomal characterization included diameter, encapsulation efficiency and stability. Minimum Inhibitory Concentration (MIC) was determined for free and liposomal forms of both drugs. Minimum Bactericidal Concentration (MBC) was determined at concentrations 1, 2 and 4 times MIC. Average diameter of liposomes was 131.88 nm and encapsulation efficiency for cefepime and ceftazidime were 2.29% end 5.77%, respectively. Improved stability was obtained when liposome formulations were prepared with a 50% molar ratio for cholesterol in relation to the phospholipid. MIC for liposomal antibiotics for both drugs were 50% lower than that of the free drug, demonstrating that liposomal drug delivery systems may contribute to increase the antibacterial activity of these drugs. PMID:24031917

  18. Draft Genome Assembly of Pseudomonas aeruginosa Quality Control Reference Strain Boston 41501.

    PubMed

    Minogue, T D; Daligault, H E; Davenport, K W; Broomall, S M; Bruce, D C; Chain, P S; Coyne, S R; Gibbons, H S; Jaissle, J; Chertkov, O; Freitas, T; Rosenzweig, C N; Xu, Y; Johnson, S L

    2014-01-01

    We present the scaffolded genome assembly of Pseudomonas aeruginosa Boston 41501, now publicly available in GenBank (JOVK00000000) in 10 contigs placed into a single scaffold. The 6.82-Mbp genome contains 66.1% G+C content and 6,295 coding sequences, including type 4 pilus and type 3 secretion system production genes. PMID:25278526

  19. Draft Genome Assembly of Pseudomonas aeruginosa Quality Control Reference Strain Boston 41501

    PubMed Central

    Minogue, T. D.; Daligault, H. E.; Davenport, K. W.; Broomall, S. M.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Gibbons, H. S.; Jaissle, J.; Chertkov, O.; Freitas, T.; Rosenzweig, C. N.; Xu, Y.

    2014-01-01

    We present the scaffolded genome assembly of Pseudomonas aeruginosa Boston 41501, now publicly available in GenBank (JOVK00000000) in 10 contigs placed into a single scaffold. The 6.82-Mbp genome contains 66.1% G+C content and 6,295 coding sequences, including type 4 pilus and type 3 secretion system production genes. PMID:25278526

  20. Draft Genome Sequence of a Pseudomonas aeruginosa Strain Able To Decompose N,N-Dimethyl Formamide

    PubMed Central

    Yan, Ming; Xu, Lin; Wei, Li; Zhang, Liting

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium, which uses a variety of organic chemicals as carbon sources. Here, we report the genome sequence of the Cu1510 isolate from wastewater containing a high concentration of N,N-dimethyl formamide. PMID:26847883

  1. [In-vitro antibiotic resistance of hospital and non-hospital strains of Pseudomonas aeruginosa].

    PubMed

    Ceddia, T; Marinucci, M C; Parravano, N

    1979-03-30

    The AA report about the resistence towards antibiotics of 42 stocks of Pseudomonas aeruginosa isolated from hospitalized patients and of 18 stocks isolated from non hospitalized patients. The most active antibiotics are Gentamicine, Neomicine and Streptomicine. Interestingly towards Tobramicine no resistence has been detected. The stocks isolated from hospitalized patients have generally shown a higher resistence. PMID:121701

  2. Sodium houttuyfonate inhibits biofilm formation and alginate biosynthesis-associated gene expression in a clinical strain of Pseudomonas aeruginosa in vitro

    PubMed Central

    WU, DA-QIANG; CHENG, HUIJUAN; DUAN, QIANGJUN; HUANG, WEIFENG

    2015-01-01

    The increasing multidrug resistance of Pseudomonas aeruginosa has become a serious public-health problem. In the present study, the inhibitory activities of sodium houttuyfonate (SH) against biofilm formation and alginate production in a clinical strain of P. aeruginosa (AH16) were investigated in vitro using crystal violet dying and standard curve methods, respectively. The cellular morphology of P. aeruginosa treated with SH was observed using a scanning electron microscope. Furthermore, reverse transcription-quantitative polymerase chain reaction was used to identify differences in the expression levels of genes associated with alginate biosynthesis as a result of the SH treatment. The results indicated that SH significantly inhibited biofilm formation, and decreased the levels of the primary biofilm constituent, alginate, in P. aeruginosa AH16 at various stages of biofilm development. In addition, scanning electron microscopy observations demonstrated that SH markedly altered the cellular morphology and biofilm structure of P. aeruginosa. Furthermore, the results from the reverse transcription-quantitative polymerase chain reaction analysis indicated that SH inhibited biofilm formation by mitigating the expression of the algD and algR genes, which are associated with alginate biosynthesis. Therefore, the present study has provided novel insights into the potent effects and underlying mechanisms of SH-induced inhibition of biofilm formation in a clinical strain of P. aeruginosa. PMID:26622388

  3. Persistent cystic fibrosis isolate Pseudomonas aeruginosa strain RP73 exhibits an under-acylated LPS structure responsible of its low inflammatory activity.

    PubMed

    Di Lorenzo, Flaviana; Silipo, Alba; Bianconi, Irene; Lore', Nicola Ivan; Scamporrino, Andrea; Sturiale, Luisa; Garozzo, Domenico; Lanzetta, Rosa; Parrilli, Michelangelo; Bragonzi, Alessandra; Molinaro, Antonio

    2015-02-01

    Pseudomonas aeruginosa, the major pathogen involved in lethal infections in cystic fibrosis (CF) population, is able to cause permanent chronic infections that can persist over the years. This ability to chronic colonize CF airways is related to a series of adaptive bacterial changes involving the immunostimulant lipopolysaccharide (LPS) molecule. The structure of LPSs isolated from several P. aeruginosa strains showed conserved features that can undergo chemical changes during the establishment of the chronic infection. In the present paper, we report the elucidation of the structure and the biological activity of the R-LPS (lipooligosaccharide, LOS) isolated from the persistent CF isolate P. aeruginosa strain RP73, in order to give further insights in the adaptation mechanism of the pathogen in the CF environment. The complete structural analysis of P. aeruginosa RP73 LOS was achieved by chemical analyses, NMR spectroscopy and MALDI MS spectrometry, while the assessment of the biological activity was attained testing the in vivo pro-inflammatory capacity of the isolated LOS molecule. While a typical CF LPS is able to trigger a high immune response and production of pro-inflammatory molecules, this P. aeruginosa RP73 LOS showed to possess a low pro-inflammatory capacity. This was possible due to a singular chemical structure possessing an under-acylated lipid A very similar to the LPS of P. aeruginosa found in chronic lung diseases such as bronchiectstasis. PMID:24856407

  4. Evidence of a plasmid-encoded oxidative xylose-catabolic pathway in Arthrobacter nicotinovorans pAO1.

    PubMed

    Mihasan, Marius; Stefan, Marius; Hritcu, Lucian; Artenie, Vlad; Brandsch, Roderich

    2013-01-01

    Due to its high abundance, the D-xylose fraction of lignocellulose provides a promising resource for production of various chemicals. Examples of efficient utilization of d-xylose are nevertheless rare, mainly due to the lack of enzymes with suitable properties for biotechnological applications. The genus Arthrobacter, which occupies an ecological niche rich in lignocellulosic materials and containing species with high resistance and tolerance to environmental factors, is a very suitable candidate for finding D-xylose-degrading enzymes with new properties. In this work, the presence of the pAO1 megaplasmid in cells of Arthrobacter nicotinovorans was directly linked to the ability of this microorganism to ferment D-xylose and to sustain longer log growth. Three pAO1 genes (orf32, orf39, orf40) putatively involved in degradation of xylose were identified and cloned, and the corresponding proteins purified and characterized. ORF40 was shown to be a homotetrameric NADP(+)/NAD(+) sugar dehydrogenase with a strong preference for d-xylose; ORF39 is a monomeric aldehyde dehydrogenase with wide substrate specificity and ORF32 is a constitutive expressed transcription factor putatively involved in control of the entire catabolic pathway. Based on analogies with other pentose degradation pathways, a putative xylose oxidative pathway similar to the Weimberg pathway is postulated. PMID:23063486

  5. Lead-enhanced siderophore production and alteration in cell morphology in a Pb-resistant Pseudomonas aeruginosa strain 4EA.

    PubMed

    Naik, Milind Mohan; Dubey, Santosh Kumar

    2011-02-01

    A lead-resistant bacterial strain 4EA from soil contaminated with car battery waste from Goa, India was isolated and identified as Pseudomonas aeruginosa. This lead-resistant bacterial isolate interestingly revealed lead-enhanced siderophore (pyochelin and pyoverdine) production up to 0.5 mM lead nitrate whereas cells exhibit a significant decline in siderophore production above 0.5 mM lead nitrate. The bacterial cells also revealed significant alteration in cell morphology as size reduction when exposed to 0.8 mM lead nitrate. Enhanced production of siderophore was evidently detected by chrome azurol S agar diffusion (CASAD) assay as increase in diameter of orange halo, and reduction in bacterial size along with significant biosorption of lead was recorded by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDX). Pseudomonas aeruginosa strain 4EA also exhibits cross tolerance to other toxic metals viz. cadmium, mercury, and zinc besides resistance to multiple antibiotics such as ampicillin, erythromycin, amikacin, cephalexin, co-trimoxazole, mecillinam, lincomycin, ciphaloridine, oleondamycin, and nalidixic acid. PMID:20661573

  6. High-resolution genotyping of Pseudomonas aeruginosa strains linked to acute post cataract surgery endophthalmitis outbreaks in India

    PubMed Central

    Kenchappa, Prashanth; Sangwan, Virender S; Ahmed, Niyaz; Rao, K Rajender; Pathengay, Avinash; Mathai, Annie; Mansoori, Tarannum; Das, Taraprasad; Hasnain, Seyed E; Sharma, Savitri

    2005-01-01

    Background Investigation of two independent outbreaks of post cataract surgery endophthalmitis identified the reservoir of epidemic strains of P. aeruginosa. Methods Patient isolates cultured from vitreous fluid of all the nine cases and from the peripheral devices of phacoemulsification machine were subjected to high-resolution Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis. Results FAFLP based genotyping of the isolates confirmed nosocomial transmission. Although biochemical characterization and antibiotic susceptibility profiles grouped all the isolates together, FAFLP based genotyping revealed that, all the outbreak isolates were derived from 2 different strains, with independent origins. One group of isolates was traced to phacoprobe and the second one to the internal tubing system of the phacoemulsification machine used in cataract surgery. In silico analysis indicated possible evolution in both the clusters of P. aeruginosa isolates due to genetic polymorphisms. The polymorphisms were mapped to gene products (cell envelope, outer membrane proteins) possibly having significant role in pathogenesis. Conclusion The present study is probably the first one to apply FAFLP typing successfully to investigate outbreaks of postoperative endophthalmitis (POE) in an ophthalmic setting, which was able to identify the source, and helped to make rational decisions on sterilization procedures that halted more cases of infection in these hospitals. PMID:16343353

  7. Characterization of carbapenem resistance mechanisms and integrons in Pseudomonas aeruginosa strains from blood samples in a French hospital.

    PubMed

    Rojo-Bezares, Beatriz; Cavalié, Laurent; Dubois, Damien; Oswald, Eric; Torres, Carmen; Sáenz, Yolanda

    2016-04-01

    Metallo-β-lactamases (MBLs), porin OprD, integrons, virulence factors and the clonal relationships were characterized in imipenem-resistant Pseudomonas aeruginosa (IRPA) isolates. Fifty-six IRPA strains were recovered from blood samples of different patients at a Toulouse teaching hospital from 2011 to 2013. Susceptibility testing of 14 antibiotics was performed by the disc diffusion method. Detection and characterization of MBLs, the oprD gene and integrons were studied by PCR and sequencing. Thirteen genes involved in the virulence of P. aeruginosa were analysed. Molecular typing of IRPA strains was performed by PFGE and multilocus sequence typing. In this study, 61 % of the IRPA isolates showed a multi-resistance phenotype. The MBL phenotype, detected in three isolates (5.4 %), was linked to the blaVIM-2 gene. The oprD gene was amplified in 55 (98.2 %) IRPA strains, and variations were observed in 54 of them. Insertion sequences (IS) truncating oprD were detected in eight IRPA strains, with the novel ISPa56 identified in two strains. Class 1 integrons were detected in 24 (42.9 %) IRPA strains. The blaVIM-2 gene was found inside the class 1 integron arrangements. The new integrons In1054 (intI1-aacA56-qacEΔ1-sul1) and In1160 (intI1-aacA4-aacC1d-ISKpn4-gcuE-qacEΔ1-sul1) have been described for the first time, to the best of our knowledge, in this study. A high clonal diversity was found in our strains. Among the variety of sequence types (STs) found, ST175, ST233, ST235, ST244 and ST654 were noteworthy as epidemic clones. In conclusion, 5.4 % of IRPA strains showed an MBL phenotype linked to the blaVIM-2 gene. The identified oprD high polymorphism could be implicated in the variable resistance to carbapenems in IRPA strains. The dissemination of high-risk clones is a cause of concern. PMID:26838942

  8. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation. PMID:27392247

  9. Pseudomonas aeruginosa d-Arabinofuranose Biosynthetic Pathway and Its Role in Type IV Pilus Assembly*

    PubMed Central

    Harvey, Hanjeong; Kus, Julianne V.; Tessier, Luc; Kelly, John; Burrows, Lori L.

    2011-01-01

    Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked d-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-d-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to d-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilAIV produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae. PMID:21676874

  10. CLONING AND EXPRESSION OF THE CATA AND CATBC GENE CLUSTERS FROM PSEUDOMONAS AERUGINOSA PAO

    EPA Science Inventory

    A 9.9-kilobase (kb) BAMIII estriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. he catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol bra...

  11. Sensitizing Pseudomonas aeruginosa to antibiotics by electrochemical disruption of membrane functions.

    PubMed

    Niepa, Tagbo H R; Snepenger, Laura M; Wang, Hao; Sivan, Shiril; Gilbert, Jeremy L; Jones, Marcus B; Ren, Dacheng

    2016-01-01

    Recently, we reported synergistic effects between 70 μA/cm(2) direct current and tobramycin in killing Pseudomonas aeruginosa PAO1 persister cells, a phenomenon we named electrochemical control of persister cells (ECCP; Niepa et al. Biomaterials 33: 7356-7365, 2012). To understand the mechanism of ECCP, the effects of electrochemical treatments mediated via stainless steel 304 and carbon electrodes on P. aeruginosa PAO1 were systematically compared using complementary approaches in this study. Electron microscopic analysis revealed that μA/cm(2) level direct current (DC) caused substantial changes in the structure and membrane integrity of P. aeruginosa PAO1 cells. DC treatments using SS304 electrodes induced cell lysis, while the same level of DC generated using carbon electrodes led to aggregation of intracellular proteins and increased permeabilization of P. aeruginosa PAO1 cells to antibiotics. The profound effects of DC on the physiology of persister cells were corroborated with DNA microarray analysis, which revealed the induction of genes associated with pyocin production and SOS response in DC-treated persister cells. Interestingly, sequential treatment using DC mediated with carbon electrodes followed by tobramycin was found more effective than concurrent treatment; and total eradication of persister cells was achieved. PMID:26461119

  12. Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients.

    PubMed Central

    Kersulyte, D; Struelens, M J; Deplano, A; Berg, D E

    1995-01-01

    Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency. PMID:7559985

  13. Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

    SciTech Connect

    Han, Seungil; Zaniewski, Richard P.; Marr, Eric S.; Lacey, Brian M.; Tomaras, Andrew P.; Evdokimov, Artem; Miller, J. Richard; Shanmugasundaram, Veerabahu

    2012-02-08

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by {beta}-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed {beta}-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

  14. Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

    PubMed

    Maspoli, Alessandro; Wenner, Nicolas; Mislin, Gaëtan L A; Reimmann, Cornelia

    2014-06-01

    Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host. PMID:24682869

  15. A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

    PubMed

    Malphettes, Laetitia; Weber, Cornelia C; El-Baba, Marie Daoud; Schoenmakers, Ronald G; Aubel, Dominique; Weber, Wilfried; Fussenegger, Martin

    2005-01-01

    We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing. PMID:16002786

  16. Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa.

    PubMed

    Muller, Jocelyn Fraga; Ghosh, Sudeshna; Ikuma, Kaoru; Stevens, Ann M; Love, Nancy G

    2015-11-01

    Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment. PMID:26403431

  17. C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

    PubMed Central

    Epp, Simone F.; Köhler, Thilo; Plésiat, Patrick; Michéa-Hamzehpour, Mehri; Frey, Joachim; Pechère, Jean-Claude

    2001-01-01

    We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid l-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem. PMID:11353625

  18. Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces.

    PubMed

    Panagea, S; Winstanley, C; Walshaw, M J; Ledson, M J; Hart, C A

    2005-02-01

    We conducted an environmental survey in the Liverpool adult cystic fibrosis (CF) centre in order to determine the extent of environmental contamination with an epidemic strain of Pseudomonas aeruginosa that colonizes most CF patients in Liverpool, and to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of this strain on dry surfaces, compared with that of other CF P. aeruginosa strains, to explore factors that might contribute to its high transmissibility. Samples were collected from staff, patients and the environment (drains, bath tubs, showers, dry surfaces, respiratory equipment and air) in the inpatient ward and outpatient clinic. P. aeruginosa strains were tested using a new polymerase chain reaction amplification assay specific for the Liverpool epidemic strain (LES). LES was isolated from patients' hands, clothes and bed linen. Environmental contamination with LES was only detected in close proximity to colonized patients (external surfaces of their respiratory equipment, and spirometry machine tubing and chair) and was short-lived. No persistent environmental reservoirs were found. LES was detected in the majority of air samples from inside patients' rooms, the ward corridor and the outpatient clinic. Survival of LES on dry surfaces was significantly longer than that for some other strains tested, but not compared with other strains shown not to be transmissible. Improved environmental survival on its own, therefore, cannot explain the high transmissibility of this epidemic strain. Our study suggests that airborne dissemination plays a significant role in patient-to-patient spread of LES, and confirms the need to segregate those patients colonized by epidemic P. aeruginosa strains from all other CF patients. PMID:15620443

  19. IDENTIFICATION OF MICROCYSTIN TOXINS FROM A STRAIN OF MICROCYSTIS AERUGINOSA BY LIQUID CHROMATOGRAPHY INTRODUCTION INTO A HYBRID LINEAR ION TRAP-FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETER

    EPA Science Inventory

    The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment i...

  20. Unusual non-fluorescent broad spectrum siderophore activity (SID EGYII) by Pseudomonas aeruginosa strain EGYII DSM 101801 and a new insight towards simple siderophore bioassay.

    PubMed

    Embaby, Amira M; Heshmat, Yasmin; Hussein, Ahmed

    2016-03-01

    Present study highlights an unusual non-fluorescent hydroxamate broad spectrum siderophore (SID EGYII) activity from Pseudomonas aeruginosa strain EGYII DSM 101801, a soil bacterial isolate, along with simple low cost effective siderophore bioassay. Detection of SID EGYII activity qualitatively was proved by masking this activity against Erwinia amylovora strain EGY1 DSM 101800, an indicator strain, in well-cut diffusion assay containing 100 µM FeCl3. SID EGYII activity was expressed quantitatively as arbitrary units [Siderophore arbitrary units (SAU)] 380 SAU/mL against E. amylovora strain EGY1 DSM 101800. Maximal SID EGYII activity was achieved upon growing P. aeruginosa strain EGYII DSM 101801 in PYB broth at 180 rpm for 24 h. SID EGYII displayed a broad spectrum antimicrobial activity against some human pathogens (i.e., Gram-positive bacteria, Gram-negative bacteria and yeasts) and a fireblight plant pathogen. Interestingly, transformants of Escherichia coli JM109 (DE3)pSID/EGYII harboring P. aeruginosa strain EGYII DSM 101801 plasmid demonstrated a perceivable antimicrobial activity against E. amylovora strain EGY1 DSM 101800. The broad spectrum antimicrobial activity of the unusual non-fluorescent SID EGYII would underpin its high potential in targeting bacterial pathogens posing probable threats to human health and agricultural economy. The present simple low cost effective bioassay is a new insight towards an alternative to the expensive cumbersome siderophore Chrome Azurol S assay. PMID:27015845

  1. The MSHA strain of Pseudomonas aeruginosa (PA-MSHA) inhibits gastric carcinoma progression by inducing M1 macrophage polarization.

    PubMed

    Wang, Changming; Hu, Zunqi; Zhu, Zhenxin; Zhang, Xin; Wei, Ziran; Zhang, Yu; Hu, Dali; Cai, Qingping

    2016-05-01

    Macrophages play crucial roles in promoting tumor development and progression. In the present study, we found that the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) was efficient in inducing M1 macrophage polarization. PA-MSHA treatment increases expression of M1-related cytokines and promotes activation of murine peritoneal macrophages (MPM). Interestingly, PA-MSHA inhibits cell proliferation and migration and induces the apoptosis of gastric carcinoma cells. These effects of PA-MSHA on M1 polarization were associated with activation of NF-κB expression. Thus, inducing polarization of M1 by PA-MSHA may be one potential strategy for inhibiting gastric carcinoma progression in mice. PMID:26662800

  2. Co-Cultures of Pseudomonas aeruginosa and Roseobacter denitrificans Reveal Shifts in Gene Expression Levels Compared to Solo Cultures

    PubMed Central

    Conway, Crystal A.; Esiobu, Nwadiuto; Lopez, Jose V.

    2012-01-01

    Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations. PMID:22566756

  3. Pyocyanin, a virulence factor produced by Pseudomonas aeruginosa, alters root development through reactive oxygen species and ethylene signaling in Arabidopsis.

    PubMed

    Ortiz-Castro, Randy; Pelagio-Flores, Ramón; Méndez-Bravo, Alfonso; Ruiz-Herrera, León Francisco; Campos-García, Jesús; López-Bucio, José

    2014-04-01

    Pyocyanin acts as a virulence factor in Pseudomonas aeruginosa, a plant and animal pathogen. In this study, we evaluated the effect of pyocyanin on growth and development of Arabidopsis seedlings. Root inoculation with P. aeruginosa PAO1 strain inhibited primary root growth in wild-type (WT) Arabidopsis seedlings. In contrast, single lasI- and double rhlI-/lasI- mutants of P. aeruginosa defective in pyocyanin production showed decreased root growth inhibition concomitant with an increased phytostimulation. Treatment with pyocyanin modulates root system architecture, inhibiting primary root growth and promoting lateral root and root hair formation without affecting meristem viability or causing cell death. These effects correlated with altered proportions of hydrogen peroxide and superoxide in root tips and with an inhibition of cell division and elongation. Mutant analyses showed that pyocyanin modulation of root growth was likely independent of auxin, cytokinin, and abscisic acid but required ethylene signaling because the Arabidopsis etr1-1, ein2-1, and ein3-1 ethylene-related mutants were less sensitive to pyocyanin-induced root stoppage and reactive oxygen species (ROS) distribution. Our findings suggest that pyocyanin is an important factor modulating the interplay between ROS production and root system architecture by an ethylene-dependent signaling. PMID:24224532

  4. Identification and functional analysis of a bacteriocin, pyocin S6, with ribonuclease activity from a Pseudomonas aeruginosa cystic fibrosis clinical isolate.

    PubMed

    Dingemans, Jozef; Ghequire, Maarten G K; Craggs, Michael; De Mot, René; Cornelis, Pierre

    2016-06-01

    S-type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor-binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore-forming activity. Pyocins S2, S3, S4, and S5 recognize TonB-dependent ferri-siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin-immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor-binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3-equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron-limiting conditions. In the S1-pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6-type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6-type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one-fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non-CF clinical isolates. PMID:26860427

  5. Interaction of Cr(VI) reduction and denitrification by strain Pseudomonas aeruginosa PCN-2 under aerobic conditions.

    PubMed

    He, Da; Zheng, Maosheng; Ma, Tao; Li, Can; Ni, Jinren

    2015-06-01

    Inhibition of efficient denitrification in presence of toxic heavy metals is one of the current problems encountered in municipal wastewater treatment plants. This paper presents how to remove hexavalent chromium (Cr(VI)) and nitrate simultaneously by the novel strain Pseudomonas aeruginosa PCN-2 under aerobic conditions. The capability of strain PCN-2 for Cr(VI) and nitrate reduction was confirmed by PCR analysis of gene ChrR, napA, nirS, cnorB, nosZ, while Cr(VI) reduction was proved via an initial single-electron transfer through Cr(V) detection using electron paramagnetic resonance. Experimental results demonstrated that Cr(VI) and nitrate reduction by strain PCN-2 was much faster at pH 8-9 and higher initial cell concentration. However, increasing Cr(VI) concentration would inhibit aerobic denitrification process and result in an significant delay of nitrate reduction or N2O accumulation, which was attributed to competition between three electron acceptors, i.e., Cr(VI), O2 and nitrate in the electron transport chain. PMID:25795449

  6. Isolation of Pseudomonas aeruginosa strains from dental office environments and units in Barretos, state of São Paulo, Brazil, and analysis of their susceptibility to antimicrobial drugs

    PubMed Central

    de Oliveira, Ana Claudia; Maluta, Renato Pariz; Stella, Ariel Eurides; Rigobelo, Everlon Cid; Marin, José Moacir; de Ávila, Fernando Antonio

    2008-01-01

    A wide variety of opportunistic pathogens has been detected in the tubing supplying water to odontological equipment, in special in the biofilm lining of these tubes. Among these pathogens, Pseudomonas aeruginosa, one of the leading causes of nosocomial infections, is frequently found in water lines supplying dental units. In the present work, 160 samples of water, and 200 fomite samples from forty dental units were collected in the city of Barretos, State of São Paulo, Brazil and evaluated between January and July, 2005. Seventy-six P. aeruginosa strains, isolated from the dental environment (5 strains) and water system (71 strains), were tested for susceptibility to six antimicrobial drugs most frequently used against P. aeruginosa infections. Susceptibility to ciprofloxacin, followed by meropenem was the predominant profile. The need for effective means of reducing the microbial burden within dental unit water lines is emphasized, and the risk of exposure and cross-infection in dental practice, in special when caused by opportunistic pathogens like P. aeruginosa, are highlighted. PMID:24031269

  7. The Effect of Infection Control Nurses on the Occurrence of Pseudomonas aeruginosa Healthcare-Acquired Infection and Multidrug-Resistant Strains in Critically-Ill Children

    PubMed Central

    Xu, Wei; He, Linxi; Liu, Chunfeng; Rong, Jian; Shi, Yongyan; Song, Wenliang; Zhang, Tao; Wang, Lijie

    2015-01-01

    Background Healthcare-acquired Pseudomonas aeruginosa (P. aeruginosa) infections in the Pediatric Intensive Care Unit (PICU), which have a high incidence, increase treatment costs and mortality, and seriously threaten the safety of critically ill children. It is essential to seek convenient and effective methods to control and prevent healthcare-acquired infections (HAIs). This research was conducted to study the effect of infection control nurses on the occurrence of P. aeruginosa HAIs and multi-drug resistance (MDR) strains in PICU. Methods The clinical data was divided into two groups, with the age ranging from 1 month to 14 years. One group of the critically ill patients(N = 3,722) was admitted to PICU from 2007 to 2010, without the management of infection control nurses. The other group of the critically ill patients (N = 3,943) was admitted to PICU from 2011 to 2013, with the management of infection control nurses. Compare the mortality, morbidity and the incidence of acquired P. aeruginosa infections to evaluate the effect of infection control nurses. Results After implementation of the post of infection control nurses, the patient's overall mortality fell from 4.81% to 3.73%. Among the patients with endotracheal intubation more than 48 hours, the incidence of endotracheal intubation-related pneumonia decreased from 44.6% to 34.32%. The mortality of patients with endotracheal intubation decreased from 16.96% to 10.17%, and the morbidity of HAIs with P. aeruginosa decreased from 1.89% to 1.07%. The mutual different rate (MDR) dropped from 67.95% to 44.23%. There were remarkable differences in these rates between the two groups (p<0.05). Conclusion Implementing the post of infection control nurses is associated with effectively reducing the HAI rate, especially the incidence and morbidity of P. aeruginosa HAIs, reducing PICU mortality, improving P. aeruginosa drug resistance. PMID:26630032

  8. Production, purification, and characterization of antifungal metabolite from Pseudomonas aeruginosa SD12, a new strain obtained from tannery waste polluted soil.

    PubMed

    Dharni, Seema; Alam, Mansoor; Kalani, Komal; Abdul-Khaliq; Samad, Abdul; Srivastava, Santosh Kumar; Patra, Dharani Dhar

    2012-05-01

    A new strain, SD12, was isolated from tannery waste polluted soil and identified as Pseudomonas aeruginosa on the basis of phenotypic traits and by comparison of 16S rRNA sequences. This bacterium exhibited broad-spectrum antagonistic activity against phytopathogenic fungi. The strain produced phosphatases, cellulases, proteases, pectinases, and HCN and also retained its ability to produce hydroxamate-type siderophore. A bioactive metabolite was isolated from P. aeruginosa SD12 and was characterized as 1-hydroxyphenazine ((1-OH-PHZ) by nuclear magnetic resonance (NMR) spectral analysis. The strain was used as a biocontrol agent against root rot and wilt disease of pyrethrum caused by Rhizoctonia solani. The stain is also reported to increase the growth and biomass of Plantago ovata. The purified compound, 1-hydroxyphenazine, also showed broad-spectrum antagonistic activity towards a range of phytopathogenic fungi, which is the first report of its kind. PMID:22561863

  9. Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis.

    PubMed Central

    Dunne, W M; Buckmire, F L

    1985-01-01

    An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity. Images PMID:3935048

  10. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment. PMID:21797737

  11. Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

    PubMed

    Beeton, M L; Alves, D R; Enright, M C; Jenkins, A T A

    2015-08-01

    The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses. PMID:26100212

  12. Multidrug-Resistant Pseudomonas aeruginosa Strain That Caused an Outbreak in a Neurosurgery Ward and Its aac(6′)-Iae Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase

    PubMed Central

    Sekiguchi, Jun-ichiro; Asagi, Tsukasa; Miyoshi-Akiyama, Tohru; Fujino, Tomoko; Kobayashi, Intetsu; Morita, Koji; Kikuchi, Yoshihiro; Kuratsuji, Tadatoshi; Kirikae, Teruo

    2005-01-01

    We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, β-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of blaIMP-1, a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6′)-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6′)-Iq. Recombinant AAC(6′)-Iae protein showed aminoglycoside 6′-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6′)-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6′)-I family and mutations in drug resistance genes. PMID:16127047

  13. Clinical Strains of Pseudomonas aeruginosa Overproducing MexAB-OprM and MexXY Efflux Pumps Simultaneously

    PubMed Central

    Llanes, Catherine; Hocquet, Didier; Vogne, Christelle; Benali-Baitich, Dounia; Neuwirth, Catherine; Plésiat, Patrick

    2004-01-01

    Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa. DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon. In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon. Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics. Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants). These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY. PMID:15105137

  14. Effects of culture conditions of Pseudomonas aeruginosa strain RB on the synthesis of CdSe nanoparticles.

    PubMed

    Ayano, Hiroyuki; Kuroda, Masashi; Soda, Satoshi; Ike, Michihiko

    2015-04-01

    Cadmium selenide (CdSe) was synthesized by Pseudomonas aeruginosa strain RB in a culture containing lactic acid as a carbon source, 1 mM selenite, and 1 mM cadmium under various conditions. High purity (1.02-1.16 of the atomic ratio of Se to Cd) and efficient synthesis of biogenic CdSe nanoparticles were observed at 25-30°C, 0.05-10 g L(-1) NaCl, and neutral pH conditions compared with other tested conditions. However, the size and shape of synthesized CdSe nanoparticles were not changed by changing culture conditions. The contents of S and Se in the particles respectively increased under alkaline and weak acidic conditions. Furthermore, high temperature (>37°C), high salinity (>10 g L(-1) NaCl), and alkaline pH affected the CdSe-synthesizing rate by strain RB. This report is the first optimizing the culture conditions for synthesizing biogenic CdSe nanoparticles in a batch processing. PMID:25454693

  15. Role of the Pseudomonas quinolone signal (PQS) in sensitising Pseudomonas aeruginosa to UVA radiation.

    PubMed

    Pezzoni, Magdalena; Meichtry, Martín; Pizarro, Ramón A; Costa, Cristina S

    2015-01-01

    One of the main stress factors that bacteria face in the environment is solar ultraviolet-A (UVA) radiation, which leads to lethal effects through oxidative damage. The aim of this work was to investigate the role of 2-heptyl-3-hydroxi-4-quinolone (the Pseudomonas quinolone signal or PQS) in the response of Pseudomonas aeruginosa to UVA radiation. PQS is an intercellular quorum sensing signal associated to membrane vesicles which, among other functions, regulates genes related to iron acquisition, forms stable complexes with iron and participates in oxidative phenomena. UVA exposure of the wild-type PAO1 strain and a pqsA mutant unable to produce PQS revealed a sensitising role for this signal. Research into the mechanism involved in this phenomenon revealed that catalase, an essential factor in the UVA defence, is not related to PQS-mediated UVA sensitivity. Absorption of UVA by PQS produced its own photo-degradation, oxidation of the probe 2',7'- dichlorodihydrofluorescein and generation of singlet oxygen and superoxide anion, suggesting that this signal could be acting as an endogenous photosensitiser. The results presented in this study could explain the high sensitivity to UVA of P. aeruginosa when compared to enteric bacteria. PMID:25535873

  16. Pel promotes symmetric, short-ranged surface attachment in P. aeruginosa

    NASA Astrophysics Data System (ADS)

    Cooley, B. J.; Thatcher, Travis; Hashmi, Sara; L'Her, Guillaume; Touhami, Ahmed; Provenzano, Daniele; Gordon, Vernita

    2013-03-01

    Bacterial biofilms are surface mounted, multicellular communities of interacting bacteria that are often associated with chronic infections that resist antibiotics and damage host tissue. Bacteria in a biofilm are bound in a matrix of polymeric materials that adhere the bacteria to the surface, give the system spatial structure, and cluster the bacteria near each other. The opportunistic human pathogen Pseudomonas aeruginosa is widely studied as a model biofilm-forming organism. The polymeric matrix of P. aeruginosa strain PAO1 biofilms is dominated by two bacteria-produced extracellular polymers, Pel and Psl. We use both optical and atomic force microscopy to examine the roles of these polymers in very early biofilm development, in the hours after initial surface attachment. In agreement with other researchers, we find that Psl mediates strong attachment to a glass surface. Unexpectedly, we find that Pel promotes symmetric attachment, in the form of the rod-shaped bacteria lying flat on the surface, independently of permanent attachment to the surface. Further, the presence of Pel makes adhesion forces more short-ranged than they are with Psl alone. We suggest that these effects may result through synergistic interactions of Pel and Psl in the polymeric matrix.

  17. Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion

    PubMed Central

    Metruccio, Matteo M. E.; Evans, David J.; Gabriel, Manal M.; Kadurugamuwa, Jagath L.; Fleiszig, Suzanne M. J.

    2016-01-01

    Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections. PMID:27375592

  18. Frequency of PER, VEB, SHV, TEM and CTX-M Genes in Resistant Strains of Pseudomonas aeruginosa Producing Extended Spectrum β-Lactamases

    PubMed Central

    Bokaeian, Mohmmad; Shahraki Zahedani, Shahram; Soltanian Bajgiran, Morteza; Ansari Moghaddam, Alireza

    2014-01-01

    Background: Pseudomonas aeruginosa is the most common pathogen causing nosocomial infections. Resistance of P. aeruginosa strains to broad-spectrum cephalosporins may be mediated by extended-spectrum β-lactamases (ESBLs). Objectives: We intended to investigate the prevalence of ESBLs and antimicrobial susceptibilities of P. aeruginosa isolated from patients in Zahedan, Iran. Materials and Methods: In this cross-sectional study, during 2012–2013, 116 P. aeruginosa isolates were collected from a teaching hospital in Zahedan, Iran. Susceptibility to eight antimicrobial agents was carried out by disk diffusion method. The ESBL producing strains were detected by combination disk test (CDT). ESBL positive isolates as well as other isolates showing minimum inhibitory concentrations (MICs) ≥ 4 μg/mL for ceftazidime, cefotaxime, ceftriaxone and aztreonam, were screened for the presence of the genes encoding blaTEM, blaSHV, blaPER-1 and blaVEB-1, by polymerase chain reaction (PCR). Results: Ciprofloxacin and piperacillin were the most efficient antipseudomonal agents. The results disclosed that 19 (16.37%) of the isolates were multidrug resistant and 8 (6.89%) were ESBL-positive. Of the 116 isolates, 30 (25.86%) were resistant to at least one of the antibiotics ceftazidime, ceftriaxone, cefotaxime or aztreonam and among these 30 (100%), 4 (13.3%), 2 (6.6%) and 2 (6.6%), amplified blaTEM, blaVEB-1, blaPER-1 and blaSHV, respectively. From the 30 TEM-positive isolates, 22 were ESBL-negative. Sequencing of the ESBL genes verified the accuracy of the PCR products. Conclusions: According to our results, blaTEM-116 was the most frequent isolated ESBL gene among the P. aeruginosa strains isolated from patients. PMID:25789123

  19. The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants.

    PubMed

    Oliver, Antonio; Baquero, Fernando; Blázquez, Jesús

    2002-03-01

    We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype. PMID:11952911

  20. Spread of integron-associated VIM-type metallo-beta-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals.

    PubMed

    Giakkoupi, P; Petrikkos, G; Tzouvelekis, L S; Tsonas, S; Legakis, N J; Vatopoulos, A C

    2003-02-01

    Fifty-eight imipenem-nonsusceptible (MIC >or= 8 microg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR. In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains. PMID:12574292

  1. Impact of multidrug resistance on the pathogenicity of Pseudomonas aeruginosa: in vitro and in vivo studies.

    PubMed

    Gómez-Zorrilla, Silvia; Juan, Carlos; Cabot, Gabriel; Camoez, Mariana; Tubau, Fe; Oliver, Antonio; Dominguez, M Angeles; Ariza, Javier; Peña, Carmen

    2016-05-01

    The biological cost of multidrug resistance in Pseudomonas aeruginosa (PA) remains unclear. This study aimed to evaluate the relationship between pathogenicity and the resistance profile of different PA strains, including the most common epidemic high-risk clones. Nine PA strains were studied, including two reference strains, PAO1 and PA14 [both susceptible to all antipseudomonals (multiS)], and seven clinical strains comprising three clinical multiS strains, a non-clonal multidrug-resistant (MDR) strain and the high-risk MDR clones ST111, ST235 and ST175. In vitro studies were performed to investigate growth rate, type III secretion system (TTSS) genotype, cytotoxicity and invasiveness. Additionally, a peritonitis/sepsis model was used in C57BL/6 mice. The in vitro bacterial duplication time was shorter in clinical multiS strains than in MDR-PA (0.42±0.08h vs. 0.55±0.14h; P=0.023). Among the clinical strains, exoU(+) genotype was observed only in the epidemic clone ST235. In the animal model, the probability of mortality at 48h was 70% for clinical multiS strains vs. 7.5% for clinical MDR-PA (P<0.001, log-rank). The high-risk clone ST235 was the only MDR strain that was able to cause mortality. Bacterial concentrations in peritoneal fluid were higher in mice inoculated with multiS strains compared with MDR-PA [log CFU/mL, 8.95 (IQR 3.42-9.32) vs. 1.98 (IQR 1.08-2.80); P<0.001]. These data indicate that MDR profiles are associated with a reduction in virulence of PA in a murine model. Further studies are needed to elucidate the clinical implications of these results. PMID:27079153

  2. Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

    PubMed Central

    Borkar, Prita S.; Bodade, Ragini G.; Rao, Srinivasa R.; Khobragade, C.N.

    2009-01-01

    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/min respectively. PMID:24031373

  3. Biosorption of uranium by Pseudomonas aeruginosa strain CSU immobilized in a novel matrix

    SciTech Connect

    Hu, M.C.Z.; Reeves, M.

    1997-01-01

    A number of polymeric materials, including calcium alginate, polyacrylamide, polysulfone, and polyurethane, were evaluated as possible immobilization matrices for lyophilized biomass of P. aeruginoso CSU. Polyurethane-based materials such as hydrogel were identified as superior candidates for biomass immobilization. A novel polyurethane gel-bead fabrication technique was developed and successfully demonstrated at pilot-plant scale for producing mass qualities of spherical, uniform-size beads. The immobilized bacterial biomass was evaluated via the measurement of sorption isotherms and dynamics within a batch, stirred-tank reactor; and loading and elution behavior within a continuous, upflow, packed-bed columnar reactor. Sorption equilibrium and dynamics in a batch stirred tank were modeled with a pore-diffusion mass transfer model, by which a pore-diffusion coefficient was determined to be approximately 2.0 x 10{sup -6} cm{sup 2}/s for uranyl ion transport through the polyurethane gel matrix. The biosorbent beads were regenerable with dilute (0.01-0.1 M) sodium carbonate solutions. Preliminary column breakthrough-elution studies indicated that P. aeruginosa CSU biomass immobilized within polyurethane gel beads was effective for removal of uranium from low-concentration, acidic wastewater. 35 refs., 9 figs., 4 tabs.

  4. Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

    PubMed

    Jeukens, Julie; Boyle, Brian; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Aaron, Shawn D; Charette, Steve J; Fothergill, Joanne L; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

    2014-01-01

    Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung. PMID:24505294

  5. BqsR/BqsS Constitute a Two-Component System That Senses Extracellular Fe(II) in Pseudomonas aeruginosa

    PubMed Central

    Kreamer, Naomi N. K.; Wilks, Jessica C.; Marlow, Jeffrey J.; Coleman, Maureen L.

    2012-01-01

    Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium best known as the predominant opportunistic pathogen infecting the lungs of cystic fibrosis patients. In this context, it is thought to form biofilms, within which locally reducing and acidic conditions can develop that favor the stability of ferrous iron [Fe(II)]. Because iron is a signal that stimulates biofilm formation, we performed a microarray study to determine whether P. aeruginosa strain PA14 exhibits a specific transcriptional response to extracellular Fe(II). Among the genes that were most upregulated in response to Fe(II) were those encoding the two-component system BqsR/BqsS, previously identified for its role in P. aeruginosa strain PAO1 biofilm decay (13); here, we demonstrate its role in extracellular Fe(II) sensing. bqsS and bqsR form an operon together with two small upstream genes, bqsP and bqsQ, and one downstream gene, bqsT. BqsR/BqsS sense extracellular Fe(II) at physiologically relevant concentrations (>10 μM) and elicit a specific transcriptional response, including its autoregulation. The sensor distinguishes between Fe(II), Fe(III), and other dipositive cations [Ca(II), Cu(II), Mg(II), Mn(II), Zn(II)] under aerobic or anaerobic conditions. The gene that is most upregulated by BqsR/BqsS, as measured by quantitative reverse transcription-PCR (qRT-PCR), is PA14_04180, which is predicted to encode a periplasmic oligonucleotide/oligosaccharide-binding domain (OB-fold) protein. Coincident with phenazine production during batch culture growth, Fe(II) becomes the majority of the total iron pool and bqsS is upregulated. The existence of a two-component system that senses Fe(II) indicates that extracellular Fe(II) is an important environmental signal for P. aeruginosa. PMID:22194456

  6. Detection and Genetic Characterization of Metallo-β-Lactamase IMP-1 and VIM-2 in Pseudomonas aeruginosa Strains From Different Hospitals in Kermanshah, Iran

    PubMed Central

    Abiri, Ramin; Mohammadi, Pantea; Shavani, Navid; Rezaei, Mansour

    2015-01-01

    Background: Pseudomonas aeruginosais a frequent nosocomial pathogen that causes severe diseases in many settings. Carbapenems, including meropenem and imipenem, are effective antibiotics against this organism. However, the use of carbapenems has been hampered by the emergence of strains resistant to carbapenemsvia different mechanisms such as the production of metallo-β-lactamases (MBLs), which hydrolyze all carbapenems. Several kinds of MBLs have been reported, among them VIM and IMP types being the most clinically significant carbapenemases. Objectives: We aimed to determine the distribution of blaVIM-2 and blaIMP-1 transferable genes encoding MBLs in P. aeruginosa isolated from three academic hospitals in Kermanshah. Patients and Methods: From 22nd June to 22nd September 2012, 225 isolates of P. aeruginosa were collected. These isolates were tested for antibiotic susceptibility with the Kirby-Bauer disk-diffusion method, and the MBLs were assessed using the imipenem-EDTA double-disk synergy test. The isolates were investigated for blaVIM-2 and blaIMP-1 genes using polymerase chain reaction. Results: Among the 225 isolates, 33.7% (76/225) and 18.1% (41/225) were resistant to imipenem and meropenem, respectively. Of the 76 imipenem-resistant P. aeruginosa strains, 45 (59.2%) were positive for MBLs, 34 (75%) strains carried the blaIMP-1 gene, and 1 (2.2%) strain carried the blaVIM-2 gene. Conclusions: Our results showed that there was a high frequency of IMP-1 positive P. aeruginosa in the different wards of the hospitals. PMID:26495110

  7. Real-time PCR based analysis of metal resistance genes in metal resistant Pseudomonas aeruginosa strain J007.

    PubMed

    Choudhary, Sangeeta; Sar, Pinaki

    2016-07-01

    A uranium (U)-resistant and -accumulating Pseudomonas aeruginosa strain was characterized to assess the response of toxic metals toward its growth and expression of metal resistance determinants. The bacterium showed MIC (minimum inhibitory concentration) values of 6, 3, and 2 mM for Zn, Cu, and Cd, respectively; with resistance phenotype conferred by periplasmic Cu sequestering copA and RND type heavy metal efflux czcA genes. Real-time PCR-based expression analysis revealed significant upregulation of both these genes upon exposure to low concentrations of metals for short duration, whereas the global stress response gene sodA encoding superoxide dismutase enzyme was upregulated only at higher metal concentrations or longer exposure time. It could also be inferred that copA and czcA are involved in providing resistance only at low metal concentrations, whereas involvement of "global stress response" phenomenon (expression of sodA) at higher metal concentration or increased exposure was evident. This study provides significant understanding of the adaptive response of bacteria surviving in metal and radionuclide contaminated environments along with the development of real-time PCR-based quantification method of using metal resistance genes as biomarker for monitoring relevant bacteria in such habitats. PMID:26662317

  8. Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Hammond, John H.; Dolben, Emily F.; Smith, T. Jarrod; Bhuju, Sabin

    2015-01-01

    ABSTRACT In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections. IMPORTANCE Pseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently

  9. Biochemical Characterization of Inducible 'Reductase' Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4.

    PubMed

    Karandikar, Rohini; Badri, Abinaya; Phale, Prashant S

    2015-09-01

    The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 μM and V max = 34-48 μmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4. PMID:26201480

  10. Functional assessment of mycosporine-like amino acids in Microcystis aeruginosa strain PCC 7806.

    PubMed

    Hu, Chenlin; Völler, Ginka; Süßmuth, Roderich; Dittmann, Elke; Kehr, Jan-Christoph

    2015-05-01

    The biological role of the widespread mycosporine-like amino acids (MAAs) in cyanobacteria is under debate. Here, we have constructed and characterized two mutants impaired in MAA biosynthesis in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806. We could identify shinorine as the sole MAA type of the strain, which is exclusively located in the extracellular matrix. Bioinformatic studies as wells as polymerase chain reaction screening revealed that the ability to produce MAAs is sporadically distributed within the genus. Growth experiments and reactive oxygen species quantification with wild-type and mutant strains did not support a role of shinorine in protection against UV or other stress conditions in M. aeruginosa PCC 7806. The shinorine content per dry weight of cells as well as transcription of the mys gene cluster was not significantly elevated in response to UV-A, UV-B or any other stress condition tested. Remarkably, both mutants exhibited pronounced morphological changes compared with the wild type. We observed an increased accumulation and an enhanced hydrophobicity of the extracellular matrix. Our study suggests that MAAs in Microcystis play a negligible role in protection against UV radiation but might be a strain-specific trait involved in extracellular matrix formation and cell-cell interaction. PMID:25059440

  11. Profile of Virulence Factors in the Multi-Drug Resistant Pseudomonas aeruginosa Strains of Human Urinary Tract Infections (UTI)

    PubMed Central

    Habibi, Asghar; Honarmand, Ramin

    2015-01-01

    Background: Putative virulence factors are responsible for the pathogenicity of UTIs caused by Pseudomonas aeruginosa (P. aeruginosa). Resistance of P. aeruginosa to commonly used antibiotics is caused by the extreme overprescription of those antibiotics. Objectives: The goal of the present study was to investigate the prevalence of virulence factors and the antibiotic resistance patterns of P. aeruginosa isolates in UTI cases in Iran. Patients and Methods: Two hundred and fifty urine samples were collected from patients who suffered from UTIs. Samples were cultured immediately, and those that were P. aeruginosa-positive were analyzed for the presence of virulence genes using polymerase chain reaction (PCR) testing. Antimicrobial susceptibility testing (AST) was performed using the disk diffusion method. Results: Of the 250 urine samples analyzed, 8 samples (3.2%) were positive for P. aeruginosa. The prevalence of P. aeruginosa in male and female patients was 2.7% and 3.5%, respectively, (P = 0.035). In patients less than 10 years old, it was 4.2%, and in patients more than 55 years old, it was 4.2%. These were the most commonly infected groups. The highest levels of resistance were seen against ampicillin (87.5%), norfloxacin (62.5%), gentamycin (62.5%), amikacin (62.5%), and aztreonam (62.5%), while the lowest were seen for meropenem (0%), imipenem (12.5%), and polymyxin B (12.5%). LasB (87.5%), pclH (75%), pilB (75%), and exoS (75%) were the most commonly detected virulence factors in the P. aeruginosa isolates. Conclusions: It is logical to first prescribe meropenem, imipenem, and polymyxin B in cases of UTIs caused by P. aeruginosa. Medical practitioners should be aware of the presence of levels of antibiotic resistance in hospitalized UTI patients in Iran. PMID:26756017

  12. Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

    PubMed

    Bañares-España, Elena; del Mar Fernández-Arjona, María; García-Sánchez, María Jesús; Hernández-López, Miguel; Reul, Andreas; Mariné, Mariona Hernández; Flores-Moya, Antonio

    2016-05-01

    The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide. PMID:26677166

  13. Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa.

    PubMed Central

    Visca, P; Ciervo, A; Orsi, N

    1994-01-01

    A gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor. Images PMID:8106324

  14. Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

    PubMed Central

    Berrazeg, M.; Jeannot, K.; Ntsogo Enguéné, Véronique Yvette; Broutin, I.; Loeffert, S.; Fournier, D.

    2015-01-01

    Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting. PMID:26248364

  15. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

    PubMed

    van 't Wout, Emily F A; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E; Clarke, Hanna J; Tommassen, Jan; Marciniak, Stefan J; Hiemstra, Pieter S

    2015-06-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  16. Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance.

    PubMed

    Braud, Armelle; Geoffroy, Valérie; Hoegy, Françoise; Mislin, Gaëtan L A; Schalk, Isabelle J

    2010-06-01

    In order to get access to iron, Pseudomonas aeruginosa strain PAO1 produces two major siderophores pyoverdine (PVD) and pyochelin (PCH). Both siderophores are able to chelate many other metals in addition to iron. However, despite this property, only iron is transported efficiently into the bacteria by the PVD and PCH uptake pathways. Growth studies with P. aeruginosa strains showed a lower sensitivity to toxic metals for the siderophore-producing strain than for the mutants unable to produce siderophores. Moreover, addition of PVD or PCH to the growth medium of a siderophore-deficient strain considerably reduced the toxicity of toxic metals present at concentrations of 100 µM in iron-limited and iron-supplemented growth conditions. Measurement by Inductively Coupled Plasma-Atomic Emission Spectrometry of the concentration of metals present in bacteria incubated with metals in the presence or absence of PVD or PCH indicated that both siderophores were able to sequester metals from the extracellular medium of the bacteria, decreasing metal diffusion into the bacteria. Pyoverdine was able to sequester Al(3+) , Co(2+) , Cu(2+) , Eu(3+) , Ni(2+) , Pb(2+) , Tb(3+) and Zn(2+) from the extracellular medium, and PCH, Al(3+) , Co(2+) , Cu(2+) , Ni(2+) , Pb(2+) and Zn(2+) . Moreover, the presence of 100 µM Cu(2+) and Ni(2+) increased PVD production by 290% and 380%, respectively, in a medium supplemented with iron. All these data suggest that PVD and PCH may contribute to P. aeruginosa resistance to heavy metals. PMID:23766115

  17. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa.

    PubMed

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-01-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms. PMID:26423356

  18. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-10-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

  19. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

    PubMed Central

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-01-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms. PMID:26423356

  20. Catabolite repression in Pseudomonas aeruginosa PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.

    PubMed

    Valentini, Martina; Lapouge, Karine

    2013-06-01

    In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at μM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased. PMID:23253107

  1. Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Plyuta, Vladimir; Zaitseva, Julia; Lobakova, Elena; Zagoskina, Natalia; Kuznetsov, Alexander; Khmel, Inessa

    2013-11-01

    In the natural environment, bacteria predominantly exist in matrix-enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3- to 7-fold under the action of vanillin and epicatechin, and 2- to 2.5-fold in the presence of 4-hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4-hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N-3-oxo-dodecanoyl-homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N-acyl-homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs. PMID:23594262

  2. Pleiotropic effects of temperature-regulated 2-OH-lauroytransferase (PA0011) on Pseudomonas aeruginosa antibiotic resistance, virulence and type III secretion system.

    PubMed

    Wang, Bobo; Li, Bo; Liang, Ying; Li, Jing; Gao, Lang; Chen, Lin; Duan, Kangmin; Shen, Lixin

    2016-02-01

    Pseudomonas aeruginosa is an important human pathogen which adapts to changing environment, such as temperature variations and entering host by regulating their gene expression. Here, we report that gene PA0011 in P. aeruginosa PAO1, which encodes a 2-OH-lauroytransferase participating in lipid A biosynthesis, is involved in carbapenem resistance and virulence in a temperature-regulated manner in PAO1. The expression of PA0011 was higher at an environment temperature (21 °C) than that at a body temperature (37 °C). The inactivation of PA0011 rendered increased antibiotic susceptibility and decreased virulence both in vivo and in vitro. The impaired integrity and the decreased stability of the outer membrane were the cause of the increased susceptibility of PAO1(Δ0011) to carbapenem and many other common antibiotics. The reduced endotoxic activity of lipopolysaccharide (LPS) contributed to the decreased virulence both at 21 °C and 37 °C in PAO1 (Δ0011). In addition, we have found that PA0011 repressed the expression of TTSS virulence factors both at transcriptional and translational levels, similar to the effect of O antigen of LPS but unlike any effect of its homologue reported in other bacteria. The effect of PA0011 on resistance to many antibiotics including carbapenem and virulence in P. aeruginosa makes it a target for novel antimicrobial therapies. PMID:26596709

  3. Characterization of the susceptibility of Pseudomonas aeruginosa to complement-mediated killing: role of antibodies to the rough lipopolysaccharide on serum-sensitive strains.

    PubMed Central

    Schiller, N L

    1988-01-01

    The mechanism of complement-mediated killing of seven serum-sensitive Pseudomonas aeruginosa strains was examined. All seven strains were sensitive to the bactericidal activity of 20% pooled normal human serum (PNHS) containing magnesium EGTA, which blocks the classical complement pathway (CCP), or 20% PNHS preheated to 50 degrees C for 20 min, which inactivates the alternative complement pathway, suggesting that either pathway was effective against these strains. However, for four of these strains, optimal killing required the function of both pathways. Preabsorption of PNHS with serum-sensitive strains dramatically reduced the killing activity of serum for the homologous strains when a concentration of 10% serum was used, implying a role for antibody in the activation of complement via the CCP. Affinity purification of antibodies to the rough lipopolysaccharide (LPS) on strain 144M resulted in a pool of antibodies which could restore all of the bactericidal activity and most of the C3 activation-deposition activity of serum which had been lost by preabsorption with 144M. Confirmation that the LPS was the target for these bactericidal antibodies was provided by demonstrating that exogenously added 144M LPS inhibited the killing activity of PNHS. These anti-144M LPS-specific antibodies were also bactericidal for the six other serum-sensitive strains examined, suggesting that all seven strains shared an antigenic determinant recognized by these anti-144M LPS-specific antibodies. Results from cross-absorption studies imply that there are bactericidal antibodies in PNHS directed to additional bacterial targets. These studies suggest that part of the bactericidal activity of PNHS is due to binding of antibodies to the rough LPS on serum-sensitive strains, initiating activation of the CCP, and that all seven strains examined shared this bactericidal antibody-binding site. PMID:3125110

  4. Antibiotic resistance pattern and evaluation of metallo-beta lactamase genes (VIM and IMP) in Pseudomonas aeruginosa strains producing MBL enzyme, isolated from patients with secondary immunodeficiency

    PubMed Central

    Shirani, Kiana; Ataei, Behrouz; Roshandel, Fardad

    2016-01-01

    Background: One of the most common causes of hospital-acquired secondary infections in hospitalized patients is Pseudomonas aeruginosa. The aim of this study is to evaluate the expression of IMP and VIM in Pseudomonas aeruginosa strains (carbapenem resistant and producer MBL enzyme) in patients with secondary immunodeficiency. Materials and Methods: In a cross sectional study, 96 patients with secondary immunodeficiency hospitalized in the Al-Zahra hospital were selected. Carbapenem resistant strains isolated and modified Hodge test was performed in order to confirm the presence of the metallo carbapenemase enzyme. Under the standard conditions they were sent to the central laboratory for investigating nosocomial infection Multiplex PCR. Results: Of 96 samples 28.1% were IMP positive, 5.2% VIM positive and 3.1% both VIM and IMP positive. The prevalence of multidrug resistance in the IMP and/or VIM negative samples was 29%, while all 5 VIM positive samples have had multidrug resistance. Also the prevalence of multi-drug resistance in IMP positive samples were 96.3% and in IMP and VIM positive samples were 100%. According to Fisher’s test, the prevalence of multi-drug resistance based on gene expression has significant difference (P < 0.001). Conclusion: Based on the results of this study it can be concluded that, a significant percentage of patients with secondary immunodeficiency that suffer nosocomial infections with multidrug resistance, especially Pseudomonas aeruginosa, are probably MBL-producing gene positive. Therefore the cause of infection should be considered in the hospital care system to identify their features, the presence of genes involved in the development of multi-drug resistance and antibiotic therapy. PMID:27563634

  5. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

    PubMed Central

    Schwarzer, Christian; Fischer, Horst; Machen, Terry E.

    2016-01-01

    Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds. PMID:27031335

  6. The Pseudomonas aeruginosa Mannose Sensitive Hamemagglutination Strain (PA-MSHA) Induces a Th1-Polarizing Phenotype by Promoting Human Dendritic Cells Maturation.

    PubMed

    Zhang, Yunyan; Wang, Hongtao; Li, Youqiang; Chen, Ke; Ye, Jinmei; Liao, Xin; Chen, Yiyang; Ran, Wei

    2014-06-01

    Pseudomonas aeruginosa mannose sensitive hamemagglutination strain (PA-MSHA) is a kind of peritrichous P. aeruginosa strain with MSHA fimbriae and has been shown to activate kinds of immunocytes. Dendritic cells (DCs) are specialized antigen-presenting cells required for the stimulating and priming CD4(+) T cells toward the T helper cell type 1 (Th1), Th2 and other different phenotypes. PA-MSHA effecting on Th1 remains an important missing link. Here we demonstrated that PA-MSHA augmented monocytes derived-dendritic cells (Mo-DCs) expression of HLA-DR, co-stimulatory and adhesion molecules, and induced Th1-promoting interleukin-12 and tumor necrosis factor α secretion, in addition, PA-MSHA treated Mo-DCs displayed lesser endocytic capacity. Furthermore, in mixed lymphocyte reactions, allostimulatory capacity of Mo-DCs was enhanced by PA-MSHA, CD4(+) T cells stimulated by PA-MSHA -activated Mo-DCs showed a Th1-polarized cytokine production, increasing secretion of IFN-γ and decreasing secretion of IL-10 and IL-4. Our findings identified PA-MSHA as an important exogenous factor that induced DCs maturation toward a Th1-promoting phenotype. PMID:25320417

  7. Comparison of the transport and deposition of Pseudomonas aeruginosa under aerobic and anaerobic conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Huixin; Zeng, Hongbo; Ulrich, Ania C.; Liu, Yang

    2016-02-01

    Laboratory-scale columns were employed to study the effect of oxygen and ionic strength on the transport of Pseudomonas aeruginosa PAO1 in porous media. In anaerobic experiments, cells were grown and transport experiments were conducted in a well-controlled anaerobic chamber. Cell surface electrokinetic potentials were measured and surface elemental composition was analyzed using X-ray photoelectron spectroscopy (XPS). Transport experimental results showed reduced travel distance of PAO1 with increased ionic strength under aerobic and anaerobic conditions, consistent with calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The deposition rates of PAO1 were significantly higher in aerobic than in anaerobic condition at higher ionic strength (10 and 100 mM), although the electrokinetic potentials were similar throughout the tested ionic strength (1, 10, and 100 mM). No difference in PAO1 deposition rate was observed at 1 mM. XPS analysis showed that variation in cell surface composition due to different growth conditions played a primary role in determining the different deposition behaviors.

  8. Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens

    PubMed Central

    Rossignol, Gaelle; Merieau, Annabelle; Guerillon, Josette; Veron, Wilfried; Lesouhaitier, Olivier; Feuilloley, Marc GJ; Orange, Nicole

    2008-01-01

    Background Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions. Results We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of P. fluorescens or the mesophilic opportunistic pathogen, Pseudomonas aeruginosa PAO1. We demonstrated the direct, and indirect – through increases in biosurfactant release – involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by P. aeruginosa. We show that changes in PlcC production have pleiotropic effects and that plcC overexpression and plcC extinction increase MFN1032 toxicity and colonization, respectively. Conclusion This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production. PMID:18973676

  9. A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

    PubMed Central

    Bijtenhoorn, Patrick; Mayerhofer, Hubert; Müller-Dieckmann, Jochen; Utpatel, Christian; Schipper, Christina; Hornung, Claudia; Szesny, Matthias; Grond, Stephanie; Thürmer, Andrea; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Dierking, Katja; Schulenburg, Hinrich; Streit, Wolfgang R.

    2011-01-01

    In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies. PMID:22046268

  10. A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

    PubMed Central

    Vergalli, Julia; de Marsac, Nicole Tandeau; Humbert, Jean-François

    2011-01-01

    The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism. PMID:21283831

  11. The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor.

    PubMed

    Elfarash, Ameer; Wei, Qing; Cornelis, Pierre

    2012-09-01

    Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697-4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI-III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway. PMID:23170226

  12. The transcriptional regulator AlgR is essential for Pseudomonas aeruginosa pathogenesis.

    PubMed

    Lizewski, Stephen E; Lundberg, Derek S; Schurr, Michael J

    2002-11-01

    Chronic Pseudomonas aeruginosa lung infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. One P. aeruginosa virulence factor unique to CF isolates is overproduction of alginate, phenotypically termed mucoidy. Mucoidy is the result of increased transcription from the algD gene and is activated by the transcriptional regulator AlgR. Mutations in algR result in a nonmucoid phenotype and loss of twitching motility. Additionally, AlgR controls transcription of algC, encoding a dual-function enzyme necessary for both lipopolysaccharide (LPS) and alginate production. Therefore, to determine the effect of algR on P. aeruginosa virulence, an algR mutant was examined for sensitivity to reactive oxygen intermediates, killing by phagocytes, systemic virulence, and the ability to maintain a murine lung infection. We found that P. aeruginosa PAO700 (algR::Gm(r)) was less lethal than PAO1, as tested in an acute septicemia infection mouse model, and was cleared more efficiently in a mouse pneumonia model. Additionally, the algR mutant (PAO700) was more sensitive to hypochlorite. However, PAO700 was more resistant to hydrogen peroxide and killed less readily in an acellular myeloperoxidase assay than PAO1. There was little difference in killing between PAO1 and PAO700 with macrophage-like J774 cells and human polymorhonuclear leukocytes. Two-dimensional gel analysis of P. aeruginosa algR mutant and wild-type protein extracts revealed 47 differentially regulated proteins, suggesting that AlgR plays both a positive role and a negative role in gene expression. Together, these results imply that AlgR is necessary for virulence and regulates genes in addition to the genes associated with alginate and LPS production and pilus function. PMID:12379685

  13. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  14. Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

    PubMed Central

    Jackson, Angelyca A.; Gross, Maegan J.; Daniels, Emily F.; Hampton, Thomas H.; Hammond, John H.; Vallet-Gely, Isabelle; Dove, Simon L.; Stanton, Bruce A.

    2013-01-01

    Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δanr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence. PMID:23667230

  15. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  16. Cytotoxicity and inflammatory potential of two Pseudomonas mosselii strains isolated from clinical samples of hospitalized patients

    PubMed Central

    2013-01-01

    Background The genus Pseudomonas includes a heterogeneous set of microorganisms that can be isolated from many different niches and nearly 100 different strains have been described. The best characterized bacterium is Pseudomonas aeruginosa which is the primary agent of opportunistic infection in humans, causing both acute and chronic infections. Other species like fluorescens, putida or mosselii have been sporadically isolated from hospitalized patients but their association with the pathology often remains unclear. Results This study focuses on the cytotoxicity and inflammatory potential of two strains of Pseudomonas mosselii (ATCC BAA-99 and MFY161) that were recently isolated from clinical samples of hospitalized patients. The behavior of these bacteria was compared to that of the well-known opportunistic pathogen P. aeruginosa PAO1. We found that P. mosselii ATCC BAA-99 and MFY161 are cytotoxic towards Caco-2/TC7 cells, have low invasive capacity, induce secretion of human β-defensin 2 (HBD-2), alter the epithelial permeability of differentiated cells and damage the F-actin cytoskeleton. Conclusions These data bring new insights into P. mosselii virulence, since this bacterium has often been neglected due to its rare occurrence in hospital. PMID:23718251

  17. Antimicrobial activity of honey of stingless bees, tiúba (Melipona fasciculata) and jandaira (Melipona subnitida) compared to the strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Tenório, Eleuza Gomes; de Jesus, Natália Rocha; Nascimento, Adenilde Ribeiro; Teles, Amanda Mara

    2015-12-01

    This study aimed to investigate the antimicrobial activity of honeys of stingless bees produced in Maranhão, tiúba (Melipona fasciculata) and jandaira (Melipona subnitida), opposite the strains of pathogenic bacteria, namely, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The honey samples were collected from different regions of Maranhão. Of the 17 samples collected, twelve samples were honey M. fasciculata and five were honey M. subnitida. We used the Kirby-Bauer method, and the technique of agar disk diffusion through the extent of inhibition in milimetros. Results were negative for all samples from M. fasciculata. However, the tests for M. subnitida demonstrated bacteriostatic halos ranging from 12 to 32,6mm.

  18. C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

    PubMed

    Blier, Anne-Sophie; Veron, Wilfried; Bazire, Alexis; Gerault, Eloïse; Taupin, Laure; Vieillard, Julien; Rehel, Karine; Dufour, Alain; Le Derf, Franck; Orange, Nicole; Hulen, Christian; Feuilloley, Marc G J; Lesouhaitier, Olivier

    2011-07-01

    Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during

  19. C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa

    PubMed Central

    Blier, Anne-Sophie; Veron, Wilfried; Bazire, Alexis; Gerault, Eloïse; Taupin, Laure; Vieillard, Julien; Rehel, Karine; Dufour, Alain; Le Derf, Franck; Orange, Nicole; Hulen, Christian; Feuilloley, Marc G. J.

    2011-01-01

    Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during

  20. Sensitive and Specific Modified Hodge Test for KPC and Metallo-Beta- Lactamase Detection in Pseudomonas aeruginosa by Use of a Novel Indicator Strain, Klebsiella pneumoniae ATCC 700603 ▿

    PubMed Central

    Pasteran, Fernando; Veliz, Omar; Rapoport, Melina; Guerriero, Leonor; Corso, Alejandra

    2011-01-01

    We evaluated the ability of the modified Hodge test to discriminate between KPC- and metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates and carbapenemase nonproducers. With Escherichia coli ATCC 25922 as the indicator strain, the MHT resulted in low sensitivity, specificity, and repeatability. Replacing the indicator strain with Klebsiella pneumoniae ATCC 700603 led to an improved performance (100%, 97%, 0%, and 100% sensitivity, specificity, indeterminate results and repeatability, respectively). PMID:22012019

  1. Nosocomial Outbreak Due to a Multiresistant Strain of Pseudomonas aeruginosa P12: Efficacy of Cefepime-Amikacin Therapy and Analysis of β-Lactam Resistance

    PubMed Central

    Dubois, Véronique; Arpin, Corinne; Melon, Monique; Melon, Bernard; Andre, Catherine; Frigo, Cécile; Quentin, Claudine

    2001-01-01

    Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains of Pseudomonas aeruginosa P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepime-amikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49- and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant P. aeruginosa P12 exhibiting at least four mechanisms of β-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime. PMID:11376037

  2. Effect of Traditional Chinese Herbal Medicine with Antiquorum Sensing Activity on Pseudomonas aeruginosa

    PubMed Central

    Zhou, Shuxin; Jiang, Yan; Zhu, Wei; Zhuang, Xiyi; Fu, Jiangyan

    2013-01-01

    Traditional Chinese herbal medicines (TCHMs) were tested for their ability of antiquorum sensing. Water extracts of Rhubarb, Fructus gardeniae, and Andrographis paniculata show antiquorumsensing activity when using Chromobacterium violaceum CV12472 as reporter; the sub-MIC concentrations of these TCHMs were tested against AHL-dependent phenotypic expressions of PAO1. Results showed significant reduction in pyocyanin pigment, protease, elastase production, and biofilm formation in PAO1 without inhibiting the bacterial growth, revealing that the QSI by the extracts is not related to static or killing effects on the bacteria. The results indicate a potential modulation of bacterial cell-cell communication, P. aeruginosa biofilm, and virulence factors by traditional Chinese herbal medicine. This study introduces not only a new mode of action for traditional Chinese herbal medicines, but also a potential new therapeutic direction for the treatment of bacterial infections, which have QSI activity and might be important in reducing virulence and pathogenicity of pathogenic bacteria. PMID:24319480

  3. The Psl economy in early P. aeruginosa biofilm development

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Tseng, Boo Shan; Jin, Fan; Gibiansky, Max; Harrison, Joe; Parsek, Matthew; Wong, Gerard

    2012-02-01

    Psl from P. aeruginosa (PAO1) is a mannose- and galactose-rich exopolysaccharide (EPS). It has been shown that Psl plays an important role in bacterial surface adhesion. Here, we examine role of Psl in controlling motility and microcolony formation during early biofilm development, by translating video microscopy movies into searchable databases of bacterial trajectories. We use a massively-parallel cell tracking algorithm to extract the full motility history of every cell in a large community. We find that at early stages of growth, P. aeruginosa motility is guided by Psl and self-organize in a manner analogous to a capitalist economic system, resulting in a power law bacterial distribution where a small number of bacteria are extremely ``rich'' in communally produced Psl. By comparing overproducers and underproducers of Psl, we find that local Psl levels determine post-division cell fates: High local Psl levels drive the formation of sessile microcolonies that grow exponentially.

  4. Production of the Pseudomonas aeruginosa neuraminidase is increased under hyperosmolar conditions and is regulated by genes involved in alginate expression.

    PubMed Central

    Cacalano, G; Kays, M; Saiman, L; Prince, A

    1992-01-01

    The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism. In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium. The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells. This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors. The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli. In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E. coli. In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF. Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract. Images PMID:1601994

  5. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

    PubMed Central

    Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

    2013-01-01

    The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. PMID:23504942

  6. Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K.

    PubMed Central

    Wong, W. Y.; Irvin, R. T.; Paranchych, W.; Hodges, R. S.

    1992-01-01

    The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence. PMID:1284654

  7. Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections.

    PubMed

    Rojo-Molinero, Estrella; Macià, María D; Rubio, Rosa; Moyà, Bartolomé; Cabot, Gabriel; López-Causapé, Carla; Pérez, José L; Cantón, Rafael; Oliver, Antonio

    2016-05-01

    Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one

  8. Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics.

    PubMed

    Arevalo-Ferro, Catalina; Hentzer, Morten; Reil, Gerold; Görg, Angelika; Kjelleberg, Staffan; Givskov, Michael; Riedel, Kathrin; Eberl, Leo

    2003-12-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the

  9. Involvement of Pseudomonas aeruginosa rhodanese in protection from cyanide toxicity.

    PubMed

    Cipollone, Rita; Frangipani, Emanuela; Tiburzi, Federica; Imperi, Francesco; Ascenzi, Paolo; Visca, Paolo

    2007-01-01

    Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a DeltarhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the DeltarhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration. PMID:17098912

  10. Evaluation of Stress-Induced Microbial Siderophore from Pseudomonas aeruginosa Strain S1 as a Potential Matrix Metalloproteinase Inhibitor in Wound Healing Applications.

    PubMed

    Thyagarajan, Sita Lakshmi; Kandhasamy, S; Ramanathan, Giriprasath; Sivagnanam, Uma Tiruchirapalli; Perumal, P T

    2016-05-01

    Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes capable of causing various inflammatory and various degenerative diseases if over-expressed. The active site of these enzymes is a zinc binding motif which binds to the specific site on the substrate and induce degradation. Hence an inhibitor is required to form a complex with zinc motif which hampers the binding ability of MMPs. To obtain novel MMPs inhibitor for wound healing, the chelating activity of siderophore from the microbial source was focused. During screening for siderophore production, strain S1 produced the highest amount of siderophore in the minimal salts medium. The isolate was confirmed as Pseudomonas aeruginosa strain S1 based on 16S rRNA gene sequencing and phylogenetic analysis. The activity of the siderophore was assayed using chrome azurol sulphonate and purified by the chromatographic techniques. The structural evidence through Fourier transform infrared and nuclear magnetic resonance spectra revealed that the isolated siderophore is a catecholate type with the distinctive characters. The positive results of calcein and fluozin-3 assays indicate that siderophore could bind to divalent metal ions, namely Fe(2+) and Zn(2+). As the siderophore compound focused on wound healing property, the in vitro studies revealed the viability of NH3T3 fibroblast cells and its efficiency in matrix modulating was confirmed through gelatin zymogram. PMID:26804794

  11. Burn sepsis: bacterial interference with Pseudomonas aeruginosa.

    PubMed

    Levenson, S M; Gruber, D K; Gruber, C; Watford, A; Seifter, E

    1981-05-01

    The pathogenicity of several strains of Pseudomonas aeruginosa for burned rats (3 degrees scald burns, 20% body surface) following topical application of the bacteria to the burn within 1 hour after burning was established. Following this, it was demonstrated that purposeful infection of such 3 degrees scald burns of rats by a strain of Ps. aeruginosa of low virulence (JB-77) protects the rats from the lethal effect of subsequent (48-hour) topical contamination of the burn by a highly virulent strain of Ps. aeruginosa (VA-134) (p less than 0.001). This finding was confirmed in a similar experiment beginning with germfree rats. When the challenge with the highly virulent Ps. aeruginosa strain was 24 hours (rather than 48 hours) after the burning and topical contamination of the burn with the low virulence strain of Ps. aeruginosa, there was little protection (p N.S.). When burned rats were given the low virulence strain of Ps. aeruginosa by gavage right after burning, there was not protection to subsequent (48 hours) challenge by topical application of the highly virulent strain of Ps. aeruginosa to the burn (11/12 vs 12/12 dying). Our finding that purposeful infection of a 3 degrees burn of rats (conventional and also germfree) by a strain of Ps. aeruginosa of low virulence protects from the lethal effect of subsequent (48-hour) topical contamination of the burn by a highly virulent strain of Ps. aeruginosa is due, we believe, to direct bacterial interference between the two strains of pseudomonas. PMID:6785444

  12. Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis

    PubMed Central

    Shirazi, Fazal; Ferreira, Jose A. G.; Stevens, David A.; Clemons, Karl V.; Kontoyiannis, Dimitrios P.

    2016-01-01

    Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation. PMID:26930399

  13. Transcriptome Analysis of the ArgR Regulon in Pseudomonas aeruginosa

    PubMed Central

    Lu, Chung-Dar; Yang, Zhe; Li, Wei

    2004-01-01

    Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration. We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P. aeruginosa. We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine. Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions. The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter. Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system. The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR. Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis. These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds. PMID:15175299

  14. Use of RSM modeling for optimizing decolorization of simulated textile wastewater by Pseudomonas aeruginosa strain ZM130 capable of simultaneous removal of reactive dyes and hexavalent chromium.

    PubMed

    Maqbool, Zahid; Hussain, Sabir; Ahmad, Tanvir; Nadeem, Habibullah; Imran, Muhammad; Khalid, Azeem; Abid, Muhammad; Martin-Laurent, Fabrice

    2016-06-01

    Remediation of colored wastewater loaded with dyes and metal ions is a matter of interest nowadays. In this study, 220 bacteria isolated from textile wastewater were tested for their potential to decolorize each of the four reactive dyes (reactive red-120, reactive black-5, reactive yellow-2, and reactive orange-16) in the presence of a mixture of four different heavy metals (Cr, Zn, Pb, Cd) commonly found in textile effluents. Among the tested bacteria, the isolate ZM130 was found to be the most efficient in decolorizing reactive dyes in the presence of the mixture of heavy metals and was identified as Pseudomonas aeruginosa strain ZM130 by 16S rRNA gene analysis. The strain ZM130 was highly effective in simultaneously removing hexavalent chromium (25 mg L(-1)) and the azo dyes (100 mg L(-1)) from the simulated wastewater even in the presence of other three heavy metals (Zn, Pb, Cd). Simultaneous removal of chromium and azo dyes ranged as 76.6-98.7 % and 51.9-91.1 %, respectively, after 180 h incubation. On the basis of quadratic polynomial equation and response surfaces given by the response surface methodology (RSM), optimal salt content, pH, carbon co-substrate content, and level of multi-metal mixtures for decolorization of reactive red-120 in a simulated textile wastewater by the strain ZM130 were predicted to be 19.8, 7.8, and 6.33 g L(-1) and a multi-metal mixture (Cr 13.10 mg L(-1), Pb 26.21 mg L(-1), Cd 13.10 mg L(-1), Zn 26.21 mg L(-1)), respectively. Moreover, the strain ZM130 also exhibited laccase and nicotinamide adenine dinucleotide (reduced)-dichlorophenolindophenol reductase (NADH-DCIP reductase) activity during the decolorization of reactive red-120. However, the laccase activity was found to be maximum in the presence of 300 mg L(-1) of the dye as compared to other concentrations. Hence, the isolation of this strain might serve as a potential bio-resource required for developing the strategies aiming at bioremediation of the

  15. Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume

    PubMed Central

    Pottier, Laurent; Huet, Joelle; Rabemanantsoa, Christian; Kiendrebeogo, Martin; Andriantsimahavandy, Abel; Rasamindrakotroka, Andry; Stévigny, Caroline; Duez, Pierre; El Jaziri, Mondher

    2015-01-01

    Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms. PMID:26186595

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

    PubMed

    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 (R(2)) 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

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

    PubMed Central

    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

  18. Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

    PubMed Central

    Elkins, James G.; Hassett, Daniel J.; Stewart, Philip S.; Schweizer, Herbert P.; McDermott, Timothy R.

    1999-01-01

    The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H2O2. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-inducible catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H2O2, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2O2

  19. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

    PubMed Central

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  20. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa.

    PubMed

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  1. Transcriptome profiling reveals links between ParS/ParR, MexEF-OprN, and quorum sensing in the regulation of adaptation and virulence in Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    Background The ParS/ParR two component regulatory system plays critical roles for multidrug resistance in Pseudomonas aeruginosa. It was demonstrated that in the presence of antimicrobials, ParR enhances bacterial survival by distinct mechanisms including activation of the mexXY efflux genes, enhancement of lipopolysaccharide modification through the arn operon, and reduction of the expression of oprD porin. Results In this study, we report on transcriptomic analyses of P. aeruginosa PAO1 wild type and parS and parR mutants growing in a defined minimal medium. Our transcriptomic analysis provides the first estimates of transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to the known effects on drug resistance genes, transcript abundances of the quorum sensing genes (rhlIR and pqsABCDE-phnAB) were higher in both parS and parR mutants. In accordance with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of the par genes also led to increased phenazine production and swarming motility, consistent with the up-regulation of the phenazine and rhamnolipid biosynthetic genes, respectively. Conclusion Our results link the ParS/ParR two component signal transduction system to MexEF-OprN and quorum sensing systems in P. aeruginosa. These results expand our understanding of the roles of the ParS/ParR system in the regulation of gene expression in P. aeruginosa, especially in the absence of antimicrobials. PMID:24034668

  2. Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance

    PubMed Central

    Akasaka, Takaaki; Tanaka, Mayumi; Yamaguchi, Akihito; Sato, Kenichi

    2001-01-01

    The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes. PMID:11451683

  3. Capsule production by Pseudomonas aeruginosa

    SciTech Connect

    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.

  4. Bio-Engineering High Performance Microbial Strains for MEOR

    SciTech Connect

    Xiangdong Fang; Qinghong Wang; Patrick Shuler

    2007-12-30

    The main objectives of this three-year research project are: (1) to employ the latest advances in genetics and bioengineering, especially Directed Protein Evolution technology, to improve the effectiveness of the microbial enhanced oil recovery (MEOR) process. (2) to improve the surfactant activity and the thermal stability of bio-surfactant systems for MEOR; and (3) to develop improved laboratory methods and tools that screen quickly candidate bio-systems for EOR. Biosurfactants have been receiving increasing attention as Enhanced Oil Recovery (EOR) agents because of their unique properties (i.e., mild production conditions, lower toxicity, and higher biodegradability) compared to their synthetic chemical counterparts. Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including EOR and bioremediation. During the three-year of the project period, we have successfully cloned the genes involved in the rhamnolipid bio-synthesis. And by using the Transposon containing Rhamnosyltransferase gene rhlAB, we engineered the new mutant strains P. aeruginosa PEER02 and E. coli TnERAB so they can produce rhamnolipid biosurfactans. We were able to produce rhamnolipds in both P. aeroginosa PAO1-RhlA- strain and P. fluorescens ATCC15453 strain, with the increase of 55 to 175 fold in rhamnolipid production comparing with wild type bacteria strain. We have also completed the first round direct evolution studies using Error-prone PCR technique and have constructed the library of RhlAB-containing Transposon to express mutant gene in heterologous hosts. Several methods, such as colorimetric agar plate assay, colorimetric spectrophotometer assay, bioactive assay and oil spreading assay have been established to detect and screen rhamnolipid production. Our engineered P. aeruginosa PEER02 strain can produce rhamnolipids with different carbon sources as substrate. Interfacial tension analysis (IFT) showed that different rhamnolipids from different

  5. [Assessment of 2 automated microdilution techniques compared to an agar dilution method in determining sensitivity to fosfomycin in strains of carbapenem-resistant Pseudomonas aeruginosa].

    PubMed

    Gil-Romero, Yolanda; Regodón-Domínguez, Marta; Wilhelmi de Cal, Isabel; López-Fabal, Fátima; Gómez-Garcés, José Luis

    2016-01-01

    Carbapenems-resistance in Pseudomonas aeruginosa isolates has been widely reported. Fosfomycin has been shown to act synergistically with other antimicrobials. The agar dilution method was approved for susceptibility testing for fosfomycin and Pseudomonas aeruginosa. However, broth microdilution methods are the basis of systems currently used in clinical microbiology laboratories. The results of this study indicate that these methods are acceptable as susceptibility testing methods for fosfomycin against these organisms. PMID:26620604

  6. Subcutaneous Injections of the Mannose-Sensitive Hemagglutination Pilus Strain of Pseudomonas aeruginosa Stimulate Host Immunity, Reduce Bladder Cancer Size and Improve Tumor Survival in Mice.

    PubMed

    Li, Tao; Yang, Li; Fu, Sheng-Jun; Xiao, Er-Long; Yuan, Xuan; Lu, Jian-Zhong; Ma, Bao-Liang; Shi, Ting-Kai; Wang, Zhi-Ping

    2015-09-01

    We wished to evaluate the effects of Pseudomonas aeruginosa (mannose-sensitive hemagglutination pilus strain, PA-MSHA) as an immunostimulating and anti-tumor agent for treatment of bladder cancer. Immunostimulating effects were assessed by the in vitro proliferation assay of murine splenic lymphocytes. Anti-tumor effects were studied in a subcutaneous tumor model established in female C57BL/6 mice using the MB49 bladder cell line. These mice received subcutaneous injections of normal saline (control group) or PA-MSHA (high, medium, or low dose, respectively, 1.6-2.0 × 10(9), 3.2- .0 × 10(8), 6.4-8.0 × 10(7) CFU/ml) twice a week for 3 weeks. Mice survival, tumor volume, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD), serum levels of TNF-α and IFN-γ, and blood CD4(+) /CD8(+) counts were the study outcomes. We observed that PA-MSHA promoted the growth of splenic lymphocytes in vitro. In the murine tumor model, PA-MSHA prolonged mice survival and reduced tumor growth. Furthermore, VEGF and MVD were also diminished by PA-MSHA. Mice that received high and medium dose of PA-MSHA had significantly higher serum levels of IFN-γ and TNF-α (days 21 and 28), and higher levels of CD4(+) /CD8(+) cells (days 21 and 28). In conclusion, PA-MSHA exerts beneficial effects on increasing proliferation of murine splenic lymphocytes in vitro and inhibits the growth of bladder tumor in a murine model. Therefore, PA-MSHA may be useful an immunostimulating and anti-tumor agent for bladder cancer therapy. PMID:25724441

  7. The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa shift peritoneal milky spot macrophages towards an M1 phenotype to dampen peritoneal dissemination.

    PubMed

    Miao, Zhi-Feng; Zhao, Ting-Ting; Miao, Feng; Wang, Zhen-Ning; Xu, Ying-Ying; Mao, Xiao-Yun; Gao, Jian; Wu, Jian-Hua; Liu, Xing-Yu; You, Yi; Xu, Hao; Xu, Hui-Mian

    2014-05-01

    Peritoneal dissemination (PD) of tumor cells is the most frequent pattern of gastric cancer recurrence and the leading cause of death. Peritoneal milky spots are deemed as the site of origin of gastric cancer PD wherein the main cellular components are macrophages. A vaccine derived from the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has exhibit strong immune modulatory properties. In the present study, we tested the hypothesis whether the PA-MSHA vaccine activated peritoneal milky spot macrophages (PMSM) in a manner that would attenuate PD. It was observed that PA-MSHA activated PMSM towards a classical activation phenotype via a toll-like receptor4/9-dependent mechanism, which increased interleukin-12 levels and promoted the expression of co-stimulatory and antigen-presenting molecules like CD80, CD86, and MHC-II (P < 0.05). In addition, PA-MSHA-treated PMSM exhibited strong nonspecific antitumor effects in both contact-dependent and contact-independent modes of action (P < 0.05). In mice treated with PA-MSHA before inoculating gastric cancer cells, we noted alleviated PD toward the untreated mice. In conclusion, our findings demonstrated that PA-MSHA can stimulate PMSM towards an M1 phenotype and that activated PMSM inhibit gastric cancer growth and PD both in vitro and in vivo. The results of the current study provide a mechanistic insight that is relevant to the potential application of PA-MSHA in the treatment of gastric cancer-mediated PD. PMID:24385384

  8. Two mechanisms of killing of Pseudomonas aeruginosa by tobramycin assessed at multiple inocula via mechanism-based modeling.

    PubMed

    Bulitta, Jürgen B; Ly, Neang S; Landersdorfer, Cornelia B; Wanigaratne, Nicholin A; Velkov, Tony; Yadav, Rajbharan; Oliver, Antonio; Martin, Lisandra; Shin, Beom Soo; Forrest, Alan; Tsuji, Brian T

    2015-04-01

    Bacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against three Pseudomonas aeruginosa strains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 10(8.9) CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens. PMID:25645838

  9. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway.

    PubMed Central

    Vander Wauven, C; Piérard, A; Kley-Raymann, M; Haas, D

    1984-01-01

    Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce. PMID:6438064

  10. Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa.

    PubMed Central

    Hirakata, Y; Kaku, M; Mizukane, R; Ishida, K; Furuya, N; Matsumoto, T; Tateda, K; Yamaguchi, K

    1992-01-01

    We evaluated several potential effects of erythromycin (EM) on host defense systems and the virulence of Pseudomonas aeruginosa. Peritoneal macrophages obtained from mice given 250 mg of EM per kg of body weight for 7 days by the intraperitoneal, intravenous, subcutaneous, or oral route produced significantly greater amounts of thymocyte-activating factors. These data suggest that EM enhances the in vivo production of cytokines, such as interleukins 1 and 6. Treatment of P. aeruginosa D4 with subinhibitory concentrations of EM enhanced the association of bacteria with murine Kupffer cells in vitro and increased bacterial clearance from the blood in mice. EM suppressed the in vitro production of exotoxin A, total protease, elastase, and phospholipase C by P. aeruginosa D4; exotoxin A production by P. aeruginosa PA-103; and total protease production by P. aeruginosa B16 and PAO1 in a generally dose-dependent manner. These data demonstrate that EM produces various effects in addition to its direct antimicrobial activity, suggesting that it has potential as an immunomodulator or bacterial virulence-suppressing agent against P. aeruginosa and other infections. PMID:1416882

  11. Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Yang, Liang; Liu, Yang; Markussen, Trine; Høiby, Niels; Tolker-Nielsen, Tim; Molin, Søren

    2011-08-01

    Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms. PMID:21595754

  12. A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa

    PubMed Central

    Jecz, Paulina; Bartosik, Aneta A.; Glabski, Krzysztof; Jagura-Burdzy, Grazyna

    2015-01-01

    Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parSnull) is viable however it demonstrates the phenotype characteristic for parAnull or parBnull mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parSnull phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parSnull mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation. PMID:25794281

  13. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

    PubMed

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  14. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  15. C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    PubMed Central

    Chua, Song Lin; Sivakumar, Krishnakumar; Rybtke, Morten; Yuan, Mingjun; Andersen, Jens Bo; Nielsen, Thomas E.; Givskov, Michael; Tolker-Nielsen, Tim; Cao, Bin; Kjelleberg, Staffan; Yang, Liang

    2015-01-01

    Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO32–) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO32– further increased P. aeruginosa biofilm formation and resistance to TeO32–. P. aeruginosa ΔsadCΔsiaD and PAO1/plac-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO32– exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO32–. Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth. PMID:25992876

  16. The Accessory Genome of Pseudomonas aeruginosa

    PubMed Central

    Kung, Vanderlene L.; Ozer, Egon A.; Hauser, Alan R.

    2010-01-01

    Summary: Pseudomonas aeruginosa strains exhibit significant variability in pathogenicity and ecological flexibility. Such interstrain differences reflect the dynamic nature of the P. aeruginosa genome, which is composed of a relatively invariable “core genome” and a highly variable “accessory genome.” Here we review the major classes of genetic elements comprising the P. aeruginosa accessory genome and highlight emerging themes in the acquisition and functional importance of these elements. Although the precise phenotypes endowed by the majority of the P. aeruginosa accessory genome have yet to be determined, rapid progress is being made, and a clearer understanding of the role of the P. aeruginosa accessory genome in ecology and infection is emerging. PMID:21119020

  17. Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa.

    PubMed

    Zhou, Su; Yin, Hua; Tang, Shaoyu; Peng, Hui; Yin, Donggao; Yang, Yixuan; Liu, Zehua; Dang, Zhi

    2016-05-01

    Proliferation of cyanobacteria in aquatic ecosystems has caused water security problems throughout the world. Our preliminary study has showed that Pseudomonas aeruginosa can inhibit the growth of cyanobacterium, Microcystis aeruginosa. In order to explore the inhibitory mechanism of P. aeruginosa on the cell growth and synthesis of intracellular substances of M. aeruginosa, concentrations of Chlorophyll-a, intracellular protein, carbohydrate, enzyme activities and ion metabolism of M. aeruginosa, were investigated. The results indicated that 83.84% algicidal efficiency of P. aeruginosa was achieved after treatment for 7 days. The strain inhibited the reproduction of M. aeruginosa by impeding the synthesis of intracellular protein and carbohydrate of cyanobacterium, and only a very small part of intracellular protein and carbohydrate was detected after exposure to P. aeruginosa for 5 days. P. aeruginosa caused the alteration of intracellular antioxidant enzyme activity of M. aeruginosa, such as catalase, peroxidase. The accumulation of malondialdehyde aggravated membrane injury after treatment for 3 days. P. aeruginosa also affected the ion metabolism of cyanobacteria. The release of Na(+) and Cl(-) was significantly enhanced while the uptake of K(+), Ca(2+), Mg(2+), NO3(-) and SO4(2)(-) decreased. Surface morphology and intracellular structure of cyanobacteria and bacterial cells changed dramatically over time as evidenced by electron microscope (SEM) and transmission electron microscope (TEM) analysis. These results revealed that the algicidal activity of P. aeruginosa was primarily due to the fermentation liquid of P. aeruginosa that impeded the synthesis of intracellular protein and carbohydrate, and damaged the cell membrane through membrane lipid peroxidation. PMID:26866757

  18. Proteomic analysis of keratitis-associated Pseudomonas aeruginosa

    PubMed Central

    Sewell, Abby; Dunmire, Jeffrey; Wehmann, Michael; Rowe, Theresa

    2014-01-01

    Purpose To compare the proteomic profile of a clinical isolate of Pseudomonas aeruginosa (P. aeruginosa) obtained from an infected cornea of a contact lens wearer and the laboratory strain P. aeruginosa ATCC 10145. Methods Antibiotic sensitivity, motility, biofilm formation, and virulence tests were performed using standard methods. Whole protein lysates were analyzed with liquid chromatography/ tandem mass spectrometry (LC-MS/MS) in triplicate, and relative protein abundances were determined with spectral counting. The G test followed by a post hoc Holm-Sidak adjustment was used for the statistical analyses to determine significance in the differential expression of proteins between the two strains. Results A total of 687 proteins were detected. One-hundred thirty-three (133) proteins were significantly different between the two strains. Among these, 13 were upregulated, and 16 were downregulated in the clinical strain compared to ATCC 10145, whereas 57 were detected only in the clinical strain. The upregulated proteins are associated with virulence and pathogenicity. Conclusions Proteins detected at higher levels in the clinical strain of P. aeruginosa were proteins known to be virulence factors. These results confirm that the keratitis-associated P. aeruginosa strain is pathogenic and expresses a higher number of virulence factors compared to the laboratory strain ATCC 10145. Identification of the protein profile of the corneal strain of P. aeruginosa in this study will aid in elucidating novel intervention strategies for reducing the burden of P. aeruginosa infection in keratitis. PMID:25221424

  19. Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

    PubMed Central

    Stapleton, M J; Jagger, K S; Warren, R L

    1984-01-01

    Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease. Images PMID:6317657

  20. Analysis of the swimming activity of Pseudomonas aeruginosa by using photonic force microscope

    NASA Astrophysics Data System (ADS)

    Chan, Chia-Han; Chang, Bo-Jui; Huang, Ying-Jung; Fan, Chia-Chieh; Peng, Hwei-Ling; Chi, Sien; Hsu, Long

    2005-08-01

    Swimming activity of flagella is a main factor of the motility of bacteria. Flagella expressed on the surface of bacterial species serve as a primary means of motility including swimming. We propose to use optical tweezers to analyze the swimming activity of bacteria. The sample bacteria in the work is Pseudomonas aeruginosa, and it is a gram-negative bacterium and often causes leading to burn wound infections, urinary-tract infections, and pneumonia. The single polar flagellum of P. aeruginosa has been demonstrated to be important virulence and colonization factor of this opportunistic pathogen. We demonstrate a gene to regulate the bacterial swimming activity in P. aeruginosa PAO1 by biological method. However, the change of flagellar morphology was not observed by electron microscopy analysis, suggesting that the gene regulates the flagellar rotation that could not be detected by biological method. PFM exhibits a spatial resolution of a few nanometers to detect the relative position of the probe at an acquisition rate over 1 MHz. By binding a probe such as a bead or a quantum dot on the flagella, we expect the rotation of the probe due to the flagella could be detected. It is expected that the study of the swimming activity of P. aeruginosa provide potent method for the pathogenic role of the flagella in P. aeruginosa.

  1. Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis

    PubMed Central

    Dusane, Devendra H.; Damare, Samir R.; Nancharaiah, Yarlagadda V.; Ramaiah, N.; Venugopalan, Vayalam P.; Kumar, Ameeta Ravi; Zinjarde, Smita S.

    2013-01-01

    Background Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. Methodology/Principal Findings B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. Conclusion/Significance We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent. PMID:23691235

  2. Developing an international Pseudomonas aeruginosa reference panel

    PubMed Central

    De Soyza, Anthony; Hall, Amanda J; Mahenthiralingam, Eshwar; Drevinek, Pavel; Kaca, Wieslaw; Drulis-Kawa, Zuzanna; Stoitsova, Stoyanka R; Toth, Veronika; Coenye, Tom; Zlosnik, James E A; Burns, Jane L; Sá-Correia, Isabel; De Vos, Daniel; Pirnay, Jean-Paul; Kidd, Timothy J; Reid, David; Manos, Jim; Klockgether, Jens; Wiehlmann, Lutz; Tümmler, Burkhard; McClean, Siobhán; Winstanley, Craig

    2013-01-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents. PMID:24214409

  3. Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa.

    PubMed

    Salvador, Daniel; Churro, Catarina; Valério, Elisabete

    2016-04-01

    Cyanobacteria are phytoplanktonic organisms widely occurring in freshwaters, being frequently associated with the production of toxins, namely microcystins (MCs). MCs are produced non-ribosomally by a multienzyme complex (mcy genes). It has been reported that environmental factors, such as light intensity, can influence toxin production. The aim of this study was to assess the influence of light intensity in the transcription of the mcyA gene and corresponding production of microcystins in toxic isolates of Planktothrix agardhii, where little is known, and compare them to Microcystis aeruginosa. For that purpose, cultures were exposed to three different light intensities (4, 20 and 30 μmol photons m(-2) s(-1)) for 18 days at 20 ± 1 °C. The growth was followed daily using absorbance readings. Samples were collected at each growth stage for cell counting, microcystins quantification and RNA extraction. The level of transcripts was quantified by RT-qPCR and the relative expression determined using 16S rDNA, gltA and rpoC1 as reference genes. The most stable reference genes in M. aeruginosa were rpoC1 and gltA, whereas in P. agardhii were 16S rDNA and gltA. There was a correspondence between the growth rate and light intensity in M. aeruginosa and P. agardhii. The growth rates for both species were lower at 4 and higher at 30 μmol photons m(-2) s(-1). Microcystin concentration per cell was similar between light intensities in M. aeruginosa and over time, while in P. agardhii it was higher in the stationary phase at 4 μmol photons m(-2) s(-1). There were differences in the expression of mcyA between the two species. In M. aeruginosa, the highest levels of expression occurred at 4 μmol photons m(-2) s(-1) in the adaptation phase, whereas for P. agardhii it was at 4μmol photons m(-2) s(-1) in the exponential growth phase. Our results indicate that the light intensities tested had distinct influences on the growth, microcystin production and mcyA expression levels

  4. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  5. Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

    PubMed

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

    Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis. PMID:26673783

  6. Comparative studies on growth and physiological responses of unicellular and colonial Microcystis aeruginosa to Acorus calamus.

    PubMed

    Zhang, S-H; Chang, J-J; Cao, J-Y; Yang, C-L

    2015-02-01

    In order to explore the growth inhibition and physiological responses of unicellular and colonial Microcystis aeruginosa during coexistence with Acorus calamus, algal densities, chlorophyll a contents, exopolysaccharide (EPS) concentrations, malondialdehyde (MDA) contents, catalase (CAT) activities, and peroxidase (POD) activities of the two algae strains were analyzed. Although the unicellular and colonial strains of M. aeruginosa were both inhibited by A. calamus, unicellular algae were more sensitive than the colonial algae. The measurement results for EPS, MDA, CAT, and POD showed that unicellular M. aeruginosa had higher levels of stress related damage than colonial strains when they were exposed to the same density of A. calamus, and the cellular defense system of colonial M. aeruginosa was stronger than that of unicellular M. aeruginosa. Natural blooms of Microcystis are typically composed of colonial forms of M. aeruginosa, therefore future efforts to control such blooms, possibly through the development of new algicides, should focus on the unique characteristics of colonial M. aeruginosa strains. PMID:25416545

  7. Bioalteration of synthetic Fe(III)-, Fe(II)-bearing basaltic glasses and Fe-free glass in the presence of the heterotrophic bacteria strain Pseudomonas aeruginosa: Impact of siderophores

    NASA Astrophysics Data System (ADS)

    Perez, Anne; Rossano, Stéphanie; Trcera, Nicolas; Huguenot, David; Fourdrin, Chloé; Verney-Carron, Aurélie; van Hullebusch, Eric D.; Guyot, François

    2016-09-01

    This study aims to evaluate the role of micro-organisms and their siderophores in the first steps of the alteration processes of basaltic glasses in aqueous media. In this regard, three different types of glasses - with or without iron, in the reduced Fe(II) or oxidized Fe(III) states - were prepared on the basis of a simplified basaltic glass composition. Control and Pseudomonas aeruginosa inoculated experiments were performed in a buffered (pH 6.5) nutrient depleted medium to stimulate the production of the pyoverdine siderophore. Results show that the presence of P. aeruginosa has an effect on the dissolution kinetics of all glasses as most of the calculated elemental release rates are increased compared to sterile conditions. Reciprocally, the composition of the glass in contact with P. aeruginosa has an impact on the bacterial growth and siderophore production. As an essential nutrient for this microbial strain, Fe notably appears to play a central role during biotic experiments. Its presence in the glass stimulates the bacterial growth and minimizes the synthesis of pyoverdine. Moreover the initial Fe2+/Fe3+ ratio in the glasses modulates this synthesis, as pyoverdine is not detected at all in the system in contact with Fe(III)-bearing glass. Finally, the dissolution rates appear to be correlated to siderophore concentrations as they increase with respect to sterile experiments in the order Fe(III)-bearing glass < Fe(II)-bearing glass < Fe-free glass. This increase is attributed to complexation reactions between siderophores and Fe or Al for Fe(II)-bearing glass or Fe-free glass, respectively. The dissolution of an Fe-free glass is significantly improved in the presence of bacteria, as initial dissolution rates are increased by a factor of 3. This study attests to the essential role of siderophores in the P. aeruginosa-promoted dissolution processes of basaltic glasses as well as to the complex relationships between the nutritional potential of the glass and

  8. Mechanical Properties of Type IV Pili in P. Aeruginosa

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Touhami, Ahmed; Scheurwater, Edie; Harvey, Hanjeong; Burrows, Lori; Dutcher, John

    2009-03-01

    Type IV pili (Tfp) are thin flexible protein filaments that extend from the cell membrane of bacteria such as Pseudomonas aeruginosa and Neisseria gonorrhoeae. The mechanical properties of Tfp are of great importance since they allow bacteria to interact with and colonize various surfaces. In the present study, we have used atomic force microscopy (AFM) for both imaging and pulling on Tfp from P. aeruginosa (PAO1) and from its PilA, PilT, and FliC mutants. A single pilus filament was mechanically stretched and the resulting force-extension profiles were fitted using the worm-like-chain (WLC) model. The statistical distributions obtained for contour length, persistence length, and number of pili per bacteria pole, were used to evaluate the mechanical properties of a single pilus and the biogenesis functions of different proteins (PilA, PilT) involved in its assembly and disassembly. Importantly, the persistence length value of ˜ 1 μm measured in the present study, which is consistent with the curvature of the pili observed in our AFM images, is significantly lower than the value of 5 μm reported earlier by Skerker et al. (1). Our results shed new light on the role of mechanical forces that mediate bacteria-surface interactions and biofilm formation. 1- J.M. Skerker and H.C. Berg, Proc. Natl. Acad. Sci. USA, 98, 6901-6904 (2001).

  9. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

    PubMed Central

    Hentzer, Morten; Wu, Hong; Andersen, Jens Bo; Riedel, Kathrin; Rasmussen, Thomas B.; Bagge, Niels; Kumar, Naresh; Schembri, Mark A.; Song, Zhijun; Kristoffersen, Peter; Manefield, Mike; Costerton, John W.; Molin, Søren; Eberl, Leo; Steinberg, Peter; Kjelleberg, Staffan; Høiby, Niels; Givskov, Michael

    2003-01-01

    Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response. PMID:12881415

  10. Glucose Starvation-Induced Dispersal of Pseudomonas aeruginosa Biofilms Is cAMP and Energy Dependent

    PubMed Central

    Huynh, Tran T.; McDougald, Diane; Klebensberger, Janosch; Al Qarni, Budoor; Barraud, Nicolas; Rice, Scott A.; Kjelleberg, Staffan; Schleheck, David

    2012-01-01

    Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival. PMID:22905180

  11. Occurrence of Pseudomonas aeruginosa in Kuwait soil.

    PubMed

    Al-Saleh, Esmaeil; Akbar, Abrar

    2015-02-01

    Environmentally ubiquitous bacteria such as Pseudomonas aeruginosa evolved mechanisms to adapt and prevail under diverse conditions. In the current investigation, strains of P. aeruginosa demonstrating high rates of crude oil utilization and tolerance to high concentrations of heavy metals were found in both crude oil-contaminated and uncontaminated sites in Kuwait, and were dominant in the contaminated sites. The incidence of P. aeruginosa in tested soils implies the definitive pattern of crude oil contamination in the selection of the bacterial population in petroleum-contaminated sites in Kuwait. Surprisingly, the unculturable P. aeruginosa in different soil samples showed significant high similarity coefficients based on 16S-RFLP analyses, implying that the unculturable fraction of existing bacterial population in environmental samples is more stable and, hence, reliable for phylogenetic studies compared to the culturable bacteria. PMID:25014900

  12. Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

    PubMed Central

    Albrecht, Mark T; Wang, Wei; Shamova, Olga; Lehrer, Robert I; Schiller, Neal L

    2002-01-01

    Background Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. Methods The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). Results The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains. Conclusion These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS. PMID:11980587

  13. Microbial degradation of quinoline and methylquinolines. [Pseudomonas aeruginosa

    SciTech Connect

    Aislabie, J.; Bej, A.K.; Hurst, H.; Rothenburger, S.; Atlas, R.M. )

    1990-02-01

    Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and Pseudomonas. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.

  14. Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection.

    PubMed

    Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra; Visca, Paolo

    2016-08-01

    Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe(3+) uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe(2+) acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe(3+) transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

  15. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    PubMed

    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. PMID:26785289

  16. Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants.

    PubMed

    Ciofu, Oana; Mandsberg, Lotte F; Bjarnsholt, Thomas; Wassermann, Tina; Høiby, Niels

    2010-04-01

    During the chronic lung infection of patients with cystic fibrosis (CF), Pseudomonas aeruginosa can survive for long periods due to adaptive evolution mediated by genetic variation. Hypermutability is considered to play an important role in this adaptive evolution and it has been demonstrated that mutator populations are amplified in the CF lung by hitchhiking with adaptive mutations. Two of the genes that are frequently mutated in isolates from chronic infection are mucA and lasR. Loss-of-function mutations in these genes determine the phenotypic switch to mucoidy and loss of quorum sensing, which are considered hallmarks of chronic virulence. The aims of our study were to investigate (1) the genetic background of the P. aeruginosa subpopulations with non-mutator, weak or strong mutator phenotype and their dynamics during the chronic lung infection, and (2) the time sequence in which the hypermutable, mucoid and quorum-sensing-negative phenotypes emerge during chronic lung infection. For these purposes the sequences of mutS, mutL, uvrD, mutT, mutY and mutM anti-mutator genes as well as of mucA and lasR were analysed in 70 sequential P. aeruginosa isolates obtained from the respiratory secretions of 10 CF patients (one to three isolates per time point). Analysis of the genetic background of the mutator phenotype showed that mutS was the most commonly affected gene followed by mutL in isolates with strong mutator phenotype. The mutT, mutY, mutM genes were affected in isolates with low fold-changes in the mutation frequencies compared to the reference strain PAO1. Isolates with non-mutator, weak or strong mutator phenotype were represented at all time points showing co-existence of these subpopulations, which suggests parallel evolution of the various mutators in the different focal niches of infection in the CF lung. Mutations in mucA and lasR occurred earlier than mutations in the anti-mutator genes, showing that hypermutability is not a prerequisite for the

  17. Wide dispersion of ST175 clone despite high genetic diversity of carbapenem-nonsusceptible Pseudomonas aeruginosa clinical strains in 16 Spanish hospitals.

    PubMed

    García-Castillo, María; Del Campo, Rosa; Morosini, María Isabel; Riera, Elena; Cabot, Gabriel; Willems, Rob; van Mansfeld, Rosa; Oliver, Antonio; Cantón, Rafael

    2011-08-01

    During the COMParative Activity of Carbapenems Testing (COMPACT) surveillance study, 448 Pseudomonas aeruginosa clinical isolates were obtained from 16 Spanish hospitals. Nonsusceptibility (EUCAST breakpoints) to imipenem (35%), meropenem (33%), and/or doripenem (33%) was observed with 175 isolates (39%). Simultaneous resistance to these three drugs was observed with 126 of the 175 isolates (72%). Except for colistin, high resistance rates were observed among noncarbapenem antibiotics. Clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE) with SpeI, discriminating 68 patterns. Multilocus sequence typing (MLST) was performed on 84 isolates representing different PFGE types and all participating hospitals. Thirty-nine sequence types (STs) could be distinguished, and of these, ST175 (48 isolates, 10 hospitals), ST646 (16 isolates, 4 hospitals), ST532 (13 isolates, 3 hospitals), and ST111 (13 isolates, 7 hospitals) were the most frequently encountered. Minimum-spanning tree analysis confirmed a wide dissemination of different clones among participant hospitals, particularly ST175. PFGE pattern comparison within the four most frequent STs revealed that ST175 isolates were relatively uniform, while ST646, ST532, and ST111 isolates were highly diverse, with almost every isolate belonging to a unique pulsotype, even when originating from the same center. The population of carbapenem-nonsusceptible P. aeruginosa isolates from 16 hospitals is highly diverse, with one ST (ST175) representing a highly conserved clone disseminated in 10 of the 16 participant hospitals. This ST175 clone should be added to the list of P. aeruginosa clones at high risk for epidemic spread, such as the Liverpool, Manchester, and Melbourne clones previously found in cystic fibrosis patients and ST235 in the nosocomial setting. PMID:21697331

  18. Novel variant (bla(VIM-4)) of the metallo-beta-lactamase gene bla(VIM-1) in a clinical strain of Pseudomonas aeruginosa.

    PubMed

    Pournaras, Spyros; Tsakris, Athanassios; Maniati, Maria; Tzouvelekis, Leonidas S; Maniatis, Antonios N

    2002-12-01

    A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene bla(VIM-4), a novel variant of bla(VIM-1), with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy. PMID:12435718

  19. Dimerization of the type IV pilin from Pseudomonas aeruginosa strain K122-4 results in increased helix stability as measured by time-resolved hydrogen-deuterium exchange

    PubMed Central

    Lento, Cristina; Wilson, Derek J.; Audette, Gerald F.

    2015-01-01

    Truncated pilin monomers from Pseudomonas aeruginosa strain K122-4 (ΔK122) have been shown to enter a monomer-dimer equilibrium in solution prior to oligomerization into protein nanotubes. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved hydrogen-deuterium exchange mass spectrometry. Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state. PMID:26798830

  20. Dimerization of the type IV pilin from Pseudomonas aeruginosa strain K122-4 results in increased helix stability as measured by time-resolved hydrogen-deuterium exchange.

    PubMed

    Lento, Cristina; Wilson, Derek J; Audette, Gerald F

    2016-01-01

    Truncated pilin monomers from Pseudomonas aeruginosa strain K122-4 (ΔK122) have been shown to enter a monomer-dimer equilibrium in solution prior to oligomerization into protein nanotubes. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved hydrogen-deuterium exchange mass spectrometry. Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state. PMID:26798830

  1. Cross-Resistance between Triclosan and Antibiotics in Pseudomonas aeruginosa Is Mediated by Multidrug Efflux Pumps: Exposure of a Susceptible Mutant Strain to Triclosan Selects nfxB Mutants Overexpressing MexCD-OprJ

    PubMed Central

    Chuanchuen, Rungtip; Beinlich, Kerry; Hoang, Tung T.; Becher, Anna; Karkhoff-Schweizer, RoxAnn R.; Schweizer, Herbert P.

    2001-01-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Δ(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  2. Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ.

    PubMed

    Chuanchuen, R; Beinlich, K; Hoang, T T; Becher, A; Karkhoff-Schweizer, R R; Schweizer, H P

    2001-02-01

    Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Delta(mexAB-oprM) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB. The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM. PMID:11158736

  3. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

    PubMed Central

    Guragain, Manita; Lenaburg, Dirk L.; Moore, Frank S.; Reutlinger, Ian; Patrauchan, Marianna A.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca2+) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca2+, we characterized Ca2+ homeostasis in P. aeruginosa PAO1 cells. By using Ca2+-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca2+ ([Ca2+]in) is 0.14±0.05 μM. In response to external Ca2+, the [Ca2+]in quickly increased at least 13 fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca2+ modulated this response. Treatment with inhibitors known to affect Ca2+ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca2+]in response, suggesting the importance of the corresponding mechanisms in Ca2+ homeostasis. To identify Ca2+ transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca2+]in homeostasis was monitored for seven Ca2+-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca2+ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca2+- induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca2+. PMID:24074964

  4. Hydnophytum formicarum Jack ethanol extract modulates quorum sensing-controlled pathogenicity in Pseudomonas aeruginosa.

    PubMed

    Hertiani, Triana; Pratiwi, Sylvia Utami Tunjung

    2015-09-01

    The discovery of new mechanism to control microbial pathogenicity by quorum sensing modulation has generated the search for quorum sensing inhibitor from natural resources. The objective of this research was to evaluate the ability of Hydnophytum formicarum Jack (Rubiaceae) ethanol extract to antagonize cell-to cell communication. Pulverized H. formicarum tuber was macerated in ethyl alcohol 96% and evaporated to yield ethanol extract. A dillution technique using Luria-Bertani (LB) medium was used to observe the capability of the extract to reduce the violacein production in Chromobacterium violaceum. Samples in two-fold dilution were prepared to obtain 2 - 0.0625 mg/mL concentration. The effects on swimming, swarming and twitching motility as well as the formation of biofilm towards Pseudomonas aeruginosa PAO1 were recorded over control. All experiments were done in triplicate. The architecture of Ps. aeruginosa biofilm treated with samples was examined by CLSM (Confocal Laser Scanning Microscopy) . Our results suggested that the ethanol extract of H. formicarum caused violacein production inhibition. Furthermore, inhibition of Ps. aeruginosa motility and biofilm formation were recorded to be significant over control in a concentration dependent manner. H. formicarum serves as a potential source for new QS-based antibacterial drugs towards Ps. aeruginosa. PMID:26408889

  5. A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions.

    PubMed

    Alves, Diana R; Perez-Esteban, P; Kot, W; Bean, J E; Arnot, T; Hansen, L H; Enright, Mark C; Jenkins, A Tobias A

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred. PMID:26347362

  6. Clonal complex Pseudomonas aeruginosa in horses.

    PubMed

    Kidd, Timothy J; Gibson, Justine S; Moss, Susan; Greer, Ristan M; Cobbold, Rowland N; Wright, John D; Ramsay, Kay A; Grimwood, Keith; Bell, Scott C

    2011-05-01

    Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated. PMID:21183294

  7. Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

    PubMed

    Niazy, Abdurahman; Hughes, Lee E

    2015-08-01

    The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 μM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate. PMID:25917504

  8. Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

    PubMed Central

    Purevdorj, B.; Costerton, J. W.; Stoodley, P.

    2002-01-01

    Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies. PMID:12200300

  9. An investigation of Pseudomonas aeruginosa biofilm growth on novel nanocellulose fibre dressings.

    PubMed

    Powell, Lydia C; Khan, Saira; Chinga-Carrasco, Gary; Wright, Chris J; Hill, Katja E; Thomas, David W

    2016-02-10

    Nanocellulose from wood is a novel biomaterial, which is highly fibrillated at the nanoscale. This affords the material a number of advantages, including self-assembly, biodegradability and the ability to absorb and retain moisture, which highlights its potential usefulness in clinical wound-dressing applications. In these in vitro studies, the wound pathogen Pseudomonas aeruginosa PAO1 was used to assess the ability of two nanocellulose materials to impair bacterial growth (<48 h). The two nanocelluloses had a relatively small fraction of residual fibres (<4%) and thus a large fraction of nanofibrils (widths <20 nm). Scanning electron microscopy and confocal laser scanning microscopy imaging demonstrated impaired biofilm growth on the nanocellulose films and increased cell death when compared to a commercial control wound dressing, Aquacel(®). Nanocellulose suspensions inhibited bacterial growth, whilst UV-vis spectrophotometry and laser profilometry also revealed the ability of nanocellulose to form smooth, translucent films. Atomic force microscopy studies of the surface properties of nanocellulose demonstrated that PAO1 exhibited markedly contrasting morphology when grown on the nanocellulose film surfaces compared to an Aquacel(®) control dressing (p<0.05). This study highlights the potential utility of these biodegradable materials, from a renewable source, for wound dressing applications in the prevention and treatment of biofilm development. PMID:26686120

  10. Suppression of fungal growth exhibited by Pseudomonas aeruginosa.

    PubMed Central

    Kerr, J R

    1994-01-01

    Three surgery patients were monitored postoperatively, with particular reference to lung infection. In each case there was a clinical impression that Pseudomonas aeruginosa suppressed the growth of Candida albicans in patients with clinically significant lung infections from whom both of these organisms were isolated from serial sputum samples. Regrowth of C. albicans after P. aeruginosa eradication occurred in two patients, despite fluconazole therapy, to which both C. albicans isolates were susceptible. In all three patients, the strain of P. aeruginosa was found to inhibit the growth of the corresponding C. albicans strain in vitro. Further in vitro susceptibility studies revealed significant inhibition by 10 strains of P. aeruginosa of 11 strains of fungi known to infect humans; these were Candida krusei, Candida keyfr, Candida guillermondii, Candida tropicalis, Candida lusitaniae, Candida parapsilosis, Candida pseudotropicalis, Candida albicans, Torulopsis glabrata, Saccharomyces cerevisiae, and Aspergillus fumigatus. PMID:8150966

  11. Surface action of gentamicin on Pseudomonas aeruginosa.

    PubMed Central

    Kadurugamuwa, J L; Clarke, A J; Beveridge, T J

    1993-01-01

    The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from

  12. Biomarkers involved in energy metabolism and oxidative stress response in the liver of Goodea gracilis Hubbs and Turner, 1939 exposed to the microcystin-producing Microcystis aeruginosa LB85 strain.

    PubMed

    Olivares Rubio, Hugo F; Martínez-Torres, M Lysset; Nájera-Martínez, Minerva; Dzul-Caamal, Ricardo; Domínguez-López, María Lilia; García-Latorre, Ethel; Vega-López, Armando

    2015-09-01

    Goodea gracilis is an endemic fish that only habitats in some water bodies of Central Mexico that are contaminated with cyanobacteria-producing microcystins (MC); however, a lack of information on this topic prevails. With the aim to generate the first approximation about the physiological changes elicited by cyanobacterium that produce MC congeners in this fish species, specimens born in the laboratory was exposed for 96 h to cell densities of 572.5, 1145, 2290, 4580, and 9160 × 10(6) cells of Microcystis aeruginosa strain LB85/L, and a set of novel endpoint related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant forces O2., H2 O2 ) in addition to biomarkers of oxidative damage and antioxidant response was evaluated in the liver. Results suggest that high inhibition of protein serine/threonine phosphatase (PP) may trigger many metabolic processes, such as those related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant O2⋅, H2 O2 , TBARS, ROOH, RC=O) as well as antioxidant (SOD, CAT, GPx) response to oxidative stress. Particularly, we observed that inhibition of LDH and PP, and H2 O2 increase and TBARS production were the key damages induced by high densities of M. aeruginosa. However, changes between aerobic and anaerobic metabolism related with ROS metabolism and ADH/LDH balance are apparently an acclimation of this fish species to exposure to cyanobacteria or their MCs. Fish species living in environments potentially contaminated with cyanobacteria or their MCs possess mechanisms of acclimation that allow them to offset the damage induced, even in the case of fish that have never been exposed to MCs. PMID:24639371

  13. From the Environment to the Host: Re-Wiring of the Transcriptome of Pseudomonas aeruginosa from 22°C to 37°C

    PubMed Central

    Bielecki, Piotr; Suárez-Diez, María; Puchałka, Jacek; Albertí, Sebastian; dos Santos, Vitor Martins; Goldberg, Joanna B.

    2014-01-01

    Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe2+ extracellular concentration at 37°C and a peak in Fe3+ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature. PMID:24587139

  14. Txc, a new type II secretion system of Pseudomonas aeruginosa strain PA7, is regulated by the TtsS/TtsR two-component system and directs specific secretion of the CbpE chitin-binding protein.

    PubMed

    Cadoret, Frédéric; Ball, Geneviève; Douzi, Badreddine; Voulhoux, Romé

    2014-07-01

    We present here the functional characterization of a third complete type II secretion system (T2SS) found in newly sequenced Pseudomonas aeruginosa strain PA7. We call this system Txc (third Xcp homolog). This system is encoded by the RGP69 region of genome plasticity found uniquely in strain PA7. In addition to the 11 txc genes, RGP69 contains two additional genes encoding a possible T2SS substrate and a predicted unorthodox sensor protein, TtsS (type II secretion sensor). We also identified a gene encoding a two-component response regulator called TtsR (type II secretion regulator), which is located upstream of the ttsS gene and just outside RGP69. We show that TtsS and TtsR constitute a new and functional two-component system that controls the production and secretion of the RGP69-encoded T2SS substrate in a Txc-dependent manner. Finally, we demonstrate that this Txc-secreted substrate binds chitin, and we therefore name it CbpE (chitin-binding protein E). PMID:24748613

  15. Txc, a New Type II Secretion System of Pseudomonas aeruginosa Strain PA7, Is Regulated by the TtsS/TtsR Two-Component System and Directs Specific Secretion of the CbpE Chitin-Binding Protein

    PubMed Central

    Cadoret, Frédéric; Ball, Geneviève; Douzi, Badreddine

    2014-01-01

    We present here the functional characterization of a third complete type II secretion system (T2SS) found in newly sequenced Pseudomonas aeruginosa strain PA7. We call this system Txc (third Xcp homolog). This system is encoded by the RGP69 region of genome plasticity found uniquely in strain PA7. In addition to the 11 txc genes, RGP69 contains two additional genes encoding a possible T2SS substrate and a predicted unorthodox sensor protein, TtsS (type II secretion sensor). We also identified a gene encoding a two-component response regulator called TtsR (type II secretion regulator), which is located upstream of the ttsS gene and just outside RGP69. We show that TtsS and TtsR constitute a new and functional two-component system that controls the production and secretion of the RGP69-encoded T2SS substrate in a Txc-dependent manner. Finally, we demonstrate that this Txc-secreted substrate binds chitin, and we therefore name it CbpE (chitin-binding protein E). PMID:24748613

  16. Bactericidal effects of antimicrobial agents on epithelial cell-associated Pseudomonas aeruginosa.

    PubMed

    Hirakata, Yoichi; Yano, Hisakazu; Arai, Kazuaki; Kitagawa, Miho; Hatta, Masumitsu; Kunishima, Hiroyuki; Kaku, Mitsuo

    2012-06-01

    It is not clear whether antipseudomonal agents can kill cell-associated bacteria within a short time. Madin-Darby canine kidney (MDCK) and A549 cells were infected with Pseudomonas aeruginosa ATCC 27853 and PAO1 and the bactericidal activity of ceftazidime, imipenem, meropenem, gentamicin, and ciprofloxacin against the organisms was investigated. In both MDCK and A549 cells, β-lactams could not kill epithelial cell-associated bacteria within 2 h. Gentamicin at concentrations ≤32 μg/ml killed more than 99% of epithelial cell-associated bacteria. Ciprofloxacin at 0.5 μg/ml killed more than 99.9% of MDCK cell-associated bacteria. Ciprofloxacin has the strongest and most rapid bactericidal activity against epithelial cell-associated bacteria, which may be explained by the combination of potent in-vitro bactericidal activity and high penetration ability into epithelial cells. PMID:22116462

  17. Pseudomonas aeruginosa Population Structure Revisited

    PubMed Central

    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

  18. Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

    PubMed Central

    Schwarzer, Christian; Fu, Zhu; Patanwala, Maria; Hum, Lauren; Lopez-Guzman, Mirielle; Illek, Beate; Kong, Weidong; Lynch, Susan V.; Machen, Terry E.

    2014-01-01

    Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis CF) patients, a process regulated by quorum sensing molecules including N-(3-oxododecanoyl)-L-homoserine lactone, C12. C12 (10–100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψmito and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψmito and increases in Cacyto like 10–50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed. PMID:22233488

  19. Clonal relatedness and conserved integron structures in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo-{beta}-lactamase from different Italian hospitals.

    PubMed

    Riccio, Maria Letizia; Pallecchi, Lucia; Docquier, Jean-Denis; Cresti, Stefania; Catania, Maria Rosaria; Pagani, Laura; Lagatolla, Cristina; Cornaglia, Giuseppe; Fontana, Roberta; Rossolini, Gian Maria

    2005-01-01

    Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons. PMID:15616282

  20. Effect of bacterial inoculation of strains of Pseudomonas aeruginosa, Alcaligenes feacalis and Bacillus subtilis on germination, growth and heavy metal (Cd, Cr, and Ni) uptake of Brassica juncea.

    PubMed

    Ndeddy Aka, Robinson Junior; Babalola, Olubukola Oluranti

    2016-01-01

    Bacterial inoculation may influence Brassica juncea growth and heavy metal (Ni, Cr, and Cd) accumulation. Three metal tolerant bacterial isolates (BCr3, BCd33, and BNi11) recovered from mine tailings, identified as Pseudomonas aeruginosa KP717554, Alcaligenes feacalis KP717561, and Bacillus subtilis KP717559 were used. The isolates exhibited multiple plant growth beneficial characteristics including the production of indole-3-acetic acid, hydrogen cyanide, ammonia, insoluble phosphate solubilization together with the potential to protect plants against fungal pathogens. Bacterial inoculation improved seeds germination of B. juncea plant in the presence of 0.1 mM Cr, Cd, and Ni, as compared to the control treatment. Compared with control treatment, soil inoculation with bacterial isolates significantly increased the amount of soluble heavy metals in soil by 51% (Cr), 50% (Cd), and 44% (Ni) respectively. Pot experiment of B. juncea grown in soil spiked with 100 mg kg(-1) of NiCl2, 100 mg kg(-1) of CdCl2, and 150 mg kg(-1) of K2Cr2O7, revealed that inoculation with metal tolerant bacteria not only protected plants against the toxic effects of heavy metals, but also increased growth and metal accumulation of plants significantly. These findings suggest that such metal tolerant, plant growth promoting bacteria are valuable tools which could be used to develop bio-inoculants for enhancing the efficiency of phytoextraction. PMID:26503637

  1. Characterization of the Medium- and Long-Chain n-Alkanes Degrading Pseudomonas aeruginosa Strain SJTD-1 and Its Alkane Hydroxylase Genes

    PubMed Central

    Liu, Huan; Xu, Jing; Liang, Rubing; Liu, Jianhua

    2014-01-01

    A gram-negative aliphatic hydrocarbon-degrading bacterium SJTD-1 isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa by comparative analyses of the 16S rRNA sequence, phenotype, and physiological features. SJTD-1 could efficiently mineralize medium- and long-chain n-alkanes (C12-C30) as its sole carbon source within seven days, showing the most optimal growth on n-hexadecane, followed by n-octadecane, and n-eicosane. In 36 h, 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely; and 2 g/L n-hexadecane was degraded to undetectable levels within 72 h. Two putative alkane-degrading genes (gene 3623 and gene 4712) were characterized and our results indicated that their gene products were rate-limiting enzymes involved in the synergetic catabolism of C12–C16 alkanes. On the basis of bioinformatics and transcriptional analysis, two P450 monooxygenases, along with a putative AlmA-like oxygenase, were examined. Genetically defective mutants lacking the characteristic alkane hydroxylase failed to degrade n-octadecane, thereby suggesting a different catalytic mechanism for the microbial transformation of alkanes with chain lengths over C18. PMID:25165808

  2. Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

    PubMed Central

    Sivaprakasam, Senthilkumar; Dhandapani, Balaji; Mahadevan, Surianarayanan

    2011-01-01

    Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1–6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value. PMID:24031785

  3. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels.

    PubMed

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C P; Huisman, Jef

    2015-11-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2. PMID:26319871

  4. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels

    PubMed Central

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C. P.

    2015-01-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter