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Sample records for aeruginosa cf isolates

  1. Draft Genome Sequences of 63 Pseudomonas aeruginosa Isolates Recovered from Cystic Fibrosis Sputum

    PubMed Central

    Spilker, Theodore

    2016-01-01

    Here, we report the draft genome sequences of 63 Pseudomonas aeruginosa isolates, recovered in culture of sputum from 15 individuals with cystic fibrosis (CF) receiving care in a single CF care center over a 13-year period. These sequences add value to studies of within-host evolution of bacterial pathogens during chronic infection. PMID:27103710

  2. Genomic Variation among Contemporary Pseudomonas aeruginosa Isolates from Chronically Infected Cystic Fibrosis Patients

    PubMed Central

    Chung, Jade C. S.; Becq, Jennifer; Fraser, Louise; Schulz-Trieglaff, Ole; Bond, Nicholas J.; Foweraker, Juliet; Bruce, Kenneth D.; Smith, Geoffrey P.

    2012-01-01

    The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates. PMID:22753054

  3. 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

  4. 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

  5. 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

  6. Phase II studies of nebulised Arikace in CF patients with Pseudomonas aeruginosa infection

    PubMed Central

    Clancy, J P; Dupont, L; Konstan, M W; Billings, J; Fustik, S; Goss, C H; Lymp, J; Minic, P; Quittner, A L; Rubenstein, R C; Young, K R; Saiman, L; Burns, J L; Govan, J R W; Ramsey, B; Gupta, R

    2013-01-01

    Rationale Arikace is a liposomal amikacin preparation for aerosol delivery with potent Pseudomonas aeruginosa killing and prolonged lung deposition. Objectives To examine the safety and efficacy of 28 days of once-daily Arikace in cystic fibrosis (CF) patients chronically infected with P aeruginosa. Methods 105 subjects were evaluated in double-blind, placebo-controlled studies. Subjects were randomised to once-daily Arikace (70, 140, 280 and 560 mg; n=7, 5, 21 and 36 subjects) or placebo (n=36) for 28 days. Primary outcomes included safety and tolerability. Secondary outcomes included lung function (forced expiratory volume at one second (FEV1)), P aeruginosa density in sputum, and the Cystic Fibrosis Quality of Life Questionnaire—Revised (CFQ-R). Results The adverse event profile was similar among Arikace and placebo subjects. The relative change in FEV1 was higher in the 560 mg dose group at day 28 (p=0.033) and at day 56 (28 days post-treatment, 0.093L±0.203 vs −0.032L±0.119; p=0.003) versus placebo. Sputum P aeruginosa density decreased >1 log in the 560 mg group versus placebo (days 14, 28 and 35; p=0.021). The Respiratory Domain of the CFQ-R increased by the Minimal Clinically Important Difference (MCID) in 67% of Arikace subjects (560 mg) versus 36% of placebo (p=0.006), and correlated with FEV1 improvements at days 14, 28 and 42 (p<0.05). An open-label extension (560 mg Arikace) for 28 days followed by 56 days off over six cycles confirmed durable improvements in lung function and sputum P aeruginosa density (n=49). Conclusions Once-daily Arikace demonstrated acute tolerability, safety, biologic activity and efficacy in patients with CF with P aeruginosa infection. PMID:23749840

  7. OligoG CF-5/20 Disruption of Mucoid Pseudomonas aeruginosa Biofilm in a Murine Lung Infection Model.

    PubMed

    Hengzhuang, Wang; Song, Zhijun; Ciofu, Oana; Onsøyen, Edvar; Rye, Philip D; Høiby, Niels

    2016-05-01

    Biofilm growth is a universal survival strategy for bacteria, providing an effective and resilient approach for survival in an otherwise hostile environment. In the context of an infection, a biofilm provides resistance and tolerance to host immune defenses and antibiotics, allowing the biofilm population to survive and thrive under conditions that would destroy their planktonic counterparts. Therefore, the disruption of the biofilm is a key step in eradicating persistent bacterial infections, as seen in many types of chronic disease. In these studies, we used both in vitro minimum biofilm eradication concentration (MBEC) assays and an in vivo model of chronic biofilm infection to demonstrate the biofilm-disrupting effects of an alginate oligomer, OligoG CF-5/20. Biofilm infections were established in mice by tracheal instillation of a mucoid clinical isolate of Pseudomonas aeruginosa embedded in alginate polymer beads. The disruption of the biofilm by OligoG CF-5/20 was observed in a dose-dependent manner over 24 h, with up to a 2.5-log reduction in CFU in the infected mouse lungs. Furthermore, in vitro assays showed that 5% OligoG CF-5/20 significantly reduced the MBEC for colistin from 512 μg/ml to 4 μg/ml after 8 h. These findings support the potential for OligoG CF-5/20 as a biofilm disruption agent which may have clinical value in reducing the microbial burden in chronic biofilm infections. PMID:26833153

  8. Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa

    PubMed Central

    Paulsson, Magnus; Singh, Birendra; Al-Jubair, Tamim; Su, Yu-Ching; Høiby, Niels; Riesbeck, Kristian

    2016-01-01

    Background Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells. Methods We screened clinical isolates from the airways of CF patients and from the bloodstream of patients with bacteremia for binding of vitronectin. Two-dimensional SDS-PAGE and a proteomic approach was used to identify vitronectin-receptors in P. aeruginosa. Results P. aeruginosa from the airways of CF patients (n=27) bound more vitronectin than bacteremic isolates (n=15, p=0.025). Porin D (OprD) was identified as a vitronectin-binding protein. A P. aeruginosa oprD transposon insertion mutant had a decreased binding to soluble and immobilized vitronectin (p ≤ 0.001). Conclusions P. aeruginosa isolates obtained from CF patients significantly bound vitronectin. Porin D was defined as a novel P. aeruginosa vitronectin-receptor, and we postulate that the Porin D-dependent interaction with vitronectin may be important for colonization. PMID:26047937

  9. Oral colonization and susceptibility testing of Pseudomonas aeruginosa oral isolates from cystic fibrosis patients.

    PubMed

    Lindemann, R A; Newman, M G; Kaufman, A K; Le, T V

    1985-01-01

    Microbial samples from the oral cavities of cystic fibrosis (C.F.) patients and 20 age-matched normal control subjects were characterized. Mucoid variant Pseudomonas aeruginosa was isolated from the tongue, buccal mucosa, and saliva of C.F. patients only. Analysis of the data suggests that the oral cavity is a potential reservoir for this organism. Aspiration and cross-contamination from this reservoir may be important in perpetuating chronic pulmonary infection in C.F. patients. Susceptibility testing was performed on 20 mucoid variant P. aeruginosa oral isolates obtained from the patients according to standardized broth dilution procedures. The in vitro antimicrobial effects of sodium fluoride, stannous fluoride, and chlorhexidine were measured. Analysis of the data suggests that clinically safe and achievable levels of chlorhexidine and stannous fluoride may be antimicrobial. PMID:3918088

  10. Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India

    PubMed Central

    Agarwal, Gunjan; Kapil, Arti; Kabra, Susheel Kumar; Das, Bimal Kumar; Dwivedi, Sada Nand

    2005-01-01

    Background Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods. Results We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2–4 genotypes and the remaining 23 patients were colonized with the single genotype. Conclusion With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF

  11. Pseudomonas aeruginosa isolates from dental unit waterlines can be divided in two distinct groups, including one displaying phenotypes similar to isolates from cystic fibrosis patients

    PubMed Central

    Ouellet, Myriam M.; Leduc, Annie; Nadeau, Christine; Barbeau, Jean; Charette, Steve J.

    2015-01-01

    Pseudomonas aeruginosa displays broad genetic diversity, giving it an astonishing capacity to adapt to a variety of environments and to infect a wide range of hosts. While many P. aeruginosa isolates of various origins have been analyzed, isolates from cystic fibrosis (CF) patients have received the most attention. Less is known about the genetic and phenotypic diversity of P. aeruginosa isolates that colonize other environments where flourishing biofilms can be found. In the present study, 29 P. aeruginosa isolates from dental unit waterlines and CF patients were collected and their genetic and phenotypes profiles were compared to determine whether environmental and clinical isolates are related. The isolates were first classified using the random amplified polymorphic DNA method. This made it possible to distribute the isolates into one clinical cluster and two environmental clusters. The isolates in the environmental cluster that were genetically closer to the clinical cluster also displayed phenotypes similar to the clinical isolates. The isolates from the second environmental cluster displayed opposite phenotypes, particularly an increased capacity to form biofilms. The isolates in this cluster were also the only ones harboring genes that encoded specific epimerases involved in the synthesis of lipopolysaccharides, which could explain their increased ability to form biofilms. In conclusion, the isolates from the dental unit waterlines could be distributed into two clusters, with some of the environmental isolates resembled the clinical isolates. PMID:25653647

  12. 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

  13. 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

  14. 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

  15. Molecular Mechanisms of Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

    PubMed Central

    Jalal, Shah; Ciofu, Oana; Høiby, Niels; Gotoh, Naomasa; Wretlind, Bengt

    2000-01-01

    Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two- to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257–261, 1998). PMID:10681343

  16. 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

  17. The B Lymphocyte Differentiation Factor (BAFF) Is Expressed in the Airways of Children with CF and in Lungs of Mice Infected with Pseudomonas aeruginosa

    PubMed Central

    Bricio-Moreno, Laura; Fothergill, Joanne L.; Southern, Kevin W.; Winstanley, Craig; Christmas, Stephen E.; Slupsky, Joseph R.; McNamara, Paul S.; Kadioglu, Aras; Flanagan, Brian F.

    2014-01-01

    Background Chronic lung infection with Pseudomonas aeruginosa remains a major cause of mortality and morbidity among individuals with CF. Expression of mediators promoting recruitment and differentiation of B cells, or supporting antibody production is poorly understood yet could be key to controlling infection. Methods BAFF was measured in BAL from children with CF, both with and without P. aeruginosa, and controls. Mice were intra-nasally infected with P. aeruginosa strain LESB65 for up to 7 days. Cellular infiltration and expression of B cell chemoattractants and B cell differentiation factor, BAFF were measured in lung tissue. Results BAFF expression was elevated in both P. aeruginosa negative and positive CF patients and in P. aeruginosa infected mice post infection. Expression of the B cell chemoattractants CXCL13, CCL19 and CCL21 increased progressively post infection. Conclusions In a mouse model, infection with P. aeruginosa was associated with elevated expression of BAFF and other B cell chemoattractants suggesting a role for airway B cell recruitment and differentiation in the local adaptive immune response to P. aeruginosa. The paediatric CF airway, irrespective of pseudomonal infection, was found to be associated with an elevated level of BAFF implying that BAFF expression is not specific to pseudomonas infection and may be a feature of the CF airway. Despite the observed presence of a potent B cell activator, chronic colonisation is common suggesting that this response is ineffective. PMID:24847941

  18. 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

  19. 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

  20. Pseudomonas aeruginosa Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro

    PubMed Central

    2010-01-01

    Background Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. Results Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. Conclusions Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung. PMID:20141637

  1. Characterization by phenotypic and genotypic methods of metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from patients with cystic fibrosis.

    PubMed

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

    2015-01-01

    Pseudomonas aeruginosa continues to be a predominant cause of infections with high intrinsic resistance to antibiotics, resulting in treatment failure. P. aeruginosa is the leading cause of respiratory infections among cystic fibrosis (CF) patients. Resistance to carbapenem antibiotics among P. aeruginosa has been reported. Thus, this study was undertaken to characterize the metallo-β-lactamase (MBL) production of P. aeruginosa by phenotypic and genotypic methods. A total of 572 sputum samples were collected from cystic fibrosis patients along with the patient demographic details in a questionnaire. In total, 217 P. aeruginosa isolates were collected and an antibiogram revealed that 159 (73.3%) and 141 (64.9%) of these colonies exhibited resistance to imipenem and meropenem, respectively. Ceftazidime and tobramycin resistance were both identified in 112 (51.6%) isolates, and resistance to piperacillin-tazobactam, gatifloxacin and netilmicin was detected in 96 (44.2%) respective samples. A total of 62 (28.6%) respective samples were resistant to cefoperazone, cefepime and ceftriaxone. The least antibiotic resistance was shown to amikacin and ceftizoxime with 51 (23.5%) and 32 (14.7%) respective colonies resistant to the antibiotics. The minimum inhibitory concentration (MIC) for imipenem revealed a reduction in the MIC values. MBL screening by the zone enhancement method using ceftazidime plus EDTA discs demonstrated that 63 (56.25%) of the colonies were positive for MBL. A total of 53 (84.1%) samples expressed blaVIM and 48 (76.1%) expressed blaIMP genes, as detected by duplex polymerase chain reaction. In conclusion, carbapenem resistance is of great clinical concern in cystic fibrosis patients with P. aeruginosa infection. Therefore, mandatory regular screening and monitoring the resistance in P. aeruginosa among CF patients is required. PMID:25323940

  2. Genotypic analysis of Pseudomonas aeruginosa isolated from ocular infection.

    PubMed

    Yamaguchi, Satoshi; Suzuki, Takashi; Kobayashi, Takeshi; Oka, Naoko; Ishikawa, Eri; Shinomiya, Hiroto; Ohashi, Yuichi

    2014-07-01

    Pseudomonas aeruginosa is the causative pathogen of keratitis, conjunctivitis, and dacryocystitis. However little is known about their clinical epidemiology in Japan. In this study we investigated the genotypic characterization and serotype of P. aeruginosa isolates from ocular infections. Thirty-four clinical P. aeruginosa isolates were characterized according to infection type, the type III secretion system (TTSS), serotype, and multilocus sequence typing (MLST). We divided the isolates into four clinical infection types as follows: Contact lens (CL)-related keratitis (CL-keratitis; 15 isolates), non CL-related keratitis (non CL-keratitis; 8 isolates), conjunctivitis (7 isolates), and dacryocystitis (4 isolates). Regarding the TTSS classification and serotyping classification, no significant differences were found among the infection types. Two clusters (I, II) and three subclusters (A, B, C) were classified according to MLST. CL-keratitis isolates with exoU positivity were clustered in II-B, and conjunctivitis was clustered in cluster I. Some linkage was found between the genetic background and CL-keratitis or conjunctivitis. PMID:24746897

  3. Colonization of CF patients' upper airways with S. aureus contributes more decisively to upper airway inflammation than P. aeruginosa.

    PubMed

    Janhsen, Wibke Katharina; Arnold, Christin; Hentschel, Julia; Lehmann, Thomas; Pfister, Wolfgang; Baier, Michael; Böer, Klas; Hünniger, Kerstin; Kurzai, Oliver; Hipler, Uta-Christina; Mainz, Jochen Georg

    2016-10-01

    In cystic fibrosis (CF) patients' airways, inflammatory processes decisively contribute to remodeling and pulmonary destruction. The aims of this study were to compare upper airway (UAW) inflammation in the context of Staphylococcus aureus and Pseudomonas aeruginosa colonization in a longitudinal setting, and to examine further factors influencing UAW inflammation. Therefore, we analyzed soluble inflammatory mediators in noninvasively obtained nasal lavage (NL) of CF patients together with microbiology, medication, and relevant clinical parameters. NL, applying 10 mL of isotonic saline per nostril, was serially performed in 74 CF patients (326 samples). Concentrations of the inflammatory mediators' interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-9, and its anti-protease TIMP-1 were quantified by bead-based multiplexed assay, neutrophil elastase (NE) via ELISA. Culture-based microbiology of the upper and lower airways (LAW), as well as serological and clinical findings, were compiled. Our results indicate that UAW colonization with S. aureus significantly impacts the concentration of all measured inflammatory mediators in NL fluid except TIMP-1, whereas these effects were not significant for P. aeruginosa. Patients with S. aureus colonization of both the UAW and LAW showed significantly increased concentrations of IL-1β, IL-6, IL-8, MMP-9, and slightly elevated concentrations of NE in NL fluid compared to non-colonized patients. This work elaborates a survey on S. aureus' virulence factors that may contribute to this underestimated pathology. Serial assessment of epithelial lining fluid by NL reveals that colonization of the UAW with S. aureus contributes more to CF airway inflammatory processes than hitherto expected. PMID:27377929

  4. 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

  5. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

    PubMed

    Teixeira, Bertinellys; Rodulfo, Hectorina; Carreño, Numirin; Guzmán, Militza; Salazar, Elsa; De Donato, Marcos

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  6. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

    PubMed Central

    TEIXEIRA, Bertinellys; RODULFO, Hectorina; CARREÑO, Numirin; GUZMÁN, Militza; SALAZAR, Elsa; DONATO, Marcos DE

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  7. Non-coding small (micro) RNAs of Pseudomonas aeruginosa isolated from clinical isolates from adult patients with cystic fibrosis.

    PubMed

    Rao, J R; Nelson, D; Moore, J E; Millar, B C; Goldsmith, C E; Rendall, J; Elborn, J S

    2010-01-01

    MicroRNAs are a class of small non-coding RNAs widely reported in eukaryotic multicellular organisms. In this study, a number of small non-coding micro (mi)RNA species in clinical isolates of prokaryote Pseudomonas aeruginosa were obtained from the sputum of adult patients with cystic fibrosis (CF) utilising a DynaExpress miRNA cloning kit, and five miRNAs of 16-47 nucleotides that were smaller than those encountered or described (80-100 nucleotides) previously in bacterial systems were described. This report presents data on these unknown cellular miRNAs cloned from P. aeruginosa isolates from CF patients. Adapting a computational miRNA prediction model that takes advantage of the highly conserved known miRNA hair pin stems regions, the results revealed that the fold structure of the microRNAs had a high homology to the recently reported human bacterial infection response (BiR)-related microRNA, mi-146, associated with the Toll-like receptor (TLR) family, which is the primary evolutionarily conserved sensors of pathogen-associated molecular patterns (PAMPs), and known to trigger host inflammatory and immune responses. PMID:20973407

  8. Carbapenem Resistance Mechanisms in Pseudomonas aeruginosa Clinical Isolates

    PubMed Central

    Pai, Hyunjoo; Kim, Jong-Won; Kim, Jungmin; Lee, Ji Hyang; Choe, Kang Won; Gotoh, Naomasa

    2001-01-01

    In order to define the contributions of the mechanisms for carbapenem resistance in clinical strains of Pseudomonas aeruginosa, we investigated the presence of OprD, the expressions of the MexAB-OprM and MexEF-OprN systems, and the production of the β-lactamases for 44 clinical strains. All of the carbapenem-resistant isolates showed the loss of or decreased levels of OprD. Three strains overexpressed the MexAB-OprM efflux system by carrying mutations in mexR. These three strains had the amino acid substitution in MexR protein, Arg (CGG) → Gln (CAG), at the position of amino acid 70. None of the isolates, however, expressed the MexEF-OprN efflux system. For the characterization of β-lactamases, at least 13 isolates were the depressed mutants, and 12 strains produced secondary β-lactamases. Based on the above resistance mechanisms, the MICs of carbapenem for the isolates were analyzed. The MICs of carbapenem were mostly determined by the expression of OprD. The MICs of meropenem were two- to four-fold increased for the isolates which overexpressed MexAB-OprM in the background of OprD loss. However, the elevated MICs of meropenem for some individual isolates could not be explained. These findings suggested that other resistance mechanisms would play a role in meropenem resistance in clinical isolates of P. aeruginosa. PMID:11158744

  9. 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

  10. Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa.

    PubMed

    Buzid, Alyah; Muimhneacháin, Eoin Ó; Reen, F Jerry; Hayes, Phyllis E; Pardo, Leticia M; Shang, Fengjun; O'Gara, Fergal; Sperry, Jonathan; Luong, John H T; Glennon, Jeremy D; McGlacken, Gerard P

    2016-09-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1-10 μM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 μM, respectively. Graphical abstract Electrochemical detection of barakacin. PMID:27473426

  11. 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

  12. 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

  13. Genotyping of Pseudomonas aeruginosa sputum and stool isolates from cystic fibrosis patients: evidence for intestinal colonization and spreading into toilets.

    PubMed Central

    Döring, G.; Bareth, H.; Gairing, A.; Wolz, C.; Botzenhart, K.

    1989-01-01

    Three hundred and fifty-eight stool and 131 sputum specimens from 40 cystic fibrosis (CF) patients and 100 toilet sinks were investigated for occurrence of Pseudomonas aeruginosa; 67% (21/31) of the patients with chronic P. aeruginosa lung infections carried the organism repeatedly in the stool but the organism was found only once in the stools of nine uninfected patients. P. aeruginosa stool carriage was correlated to high P. aeruginosa numbers in patients' sputa. Typing of P. aeruginosa with a DNA probe showed identity of sputum and stool strains. Seven patients repeatedly carried additional stool strains, not found in the sputum, suggesting intestinal colonization. No differences were seen in the clinical state of patients with P. aeruginosa-negative stool samples and patients with positive stool samples. Toilets in households of P. aeruginosa-infected CF patients were significantly more often contaminated with P. aeruginosa (42%) than toilets in households of non-infected CF patients (20%; P less than 0.03). The study shows that P. aeruginosa-infected CF patients may harbour the organisms also in the intestinal tract, and may spread the bacteria into toilets. Images Fig. 1 PMID:2514111

  14. 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

  15. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel

    PubMed Central

    Arivett, Brock A.; Ream, Dave C.; Fiester, Steven E.; Kidane, Destaalem

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  16. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel.

    PubMed

    Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  17. Phenotypic and Genetic Characterization of Carbapenemase and ESBLs Producing Gram-negative Bacteria (GNB) Isolated from Patients with Cystic Fibrosis (CF) in Tehran Hospitals

    PubMed Central

    Vali, Parisa; Shahcheraghi, Fereshteh; Seyfipour, Maryam; Zamani, Maryam Alsadat; Allahyar, Mohammad Reza; Feizabadi, Mohammad Mehdi

    2014-01-01

    Background: Cystic Fibrosis (CF) is an autosomal recessive genetic disorder in white populations caused by mutation in a gene that encodes Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Since frequent respiratory tract infections are the major problem in patients with CF, obligation to identify the causative bacteria and determining their antibiotic resistance pattern is crucial. The purpose of this project was to detect Gram-negative bacteria (GNB) isolated from sputa of CF patients and to determine their antibiotic resistance pattern. Materials and Methods: The sputum of 52 CF patients, treated as inpatients at hospitals in Tehran, was obtained between November 2011 and June 2012. Samples cultured in selective and non-selective media and GNB recognized by biochemical tests. Antimicrobial susceptibility testing to cephalosporins, aminoglycosides and carbapenems was performed by disk diffusion method and MICs of them were measured. For phenotypic detection of carbapenemase and ESBLs production, the Modified Hodge test, double disk synergy test and the combined disk methods were performed. Subsequently, the genes encoding the extended spectrum beta-lactamases (blaPER, blaCTX-M) and carbapenemases (blaIMP-1, blaGES, blaKPC, blaNDM, blaVIM-1, blaVIM-2, blaSPM, blaSIM) in Gram negative bacteria were targeted among the resistant isolates by using PCR. PFGE was used to determine any genetic relationship among the Pseudomonas aeruginosa isolated from these patients. Results: Fifty five GNB were isolated from 52 sputum samples including Pseudomonas aeruginosa, Klebsiella ozaenae, Alcaligenes xylosoxidans, Achromobacter denitrificans, Klebsiella pneumonia and Stenotrophomonas maltophilia. The rates of resistance to different antibiotic were as follows: cefixime (%80), ceftriaxone (%43), ceftazidime (%45) and meropenem (%7). The prevalence of genes encoding the ESBLs and Carbapenemases among the the phenotypically positive strains were as follows: bla

  18. Antimicrobial susceptibilities and bacteriological characteristics of bovine Pseudomonas aeruginosa and Serratia marcescens isolates from mastitis.

    PubMed

    Ohnishi, Mamoru; Sawada, Takuo; Hirose, Kazuhiko; Sato, Reiichiro; Hayashimoto, Mizuki; Hata, Eiji; Yonezawa, Chizuko; Kato, Hajime

    2011-12-29

    The presence of metallo-β-lactamase (MBL)-producing and multidrug-resistant Pseudomonas aeruginosa (MDRP) strains among bovine isolates of Gram-negative bacilli, and O-serotypes of bovine Serratia marcescens and P. aeruginosa isolates have been reported rarely. The aims of this study were to (1) elucidate antimicrobial susceptibilities and O-serotypes of P. aeruginosa and S. marcescens isolates from bovine mastitis and the presence of MBL-producers and MDRP strains among them and (2) evaluate their relationships to human isolates. We investigated the MICs of 24 antimicrobials and O-serotypes for 116 P. aeruginosa and 55 S. marcescens isolates in Japan, primarily in 2006. A total of 171 isolates exhibited high antimicrobial susceptibilities with the exception of a partial drug. P. aeruginosa isolates exhibited high susceptibilities of ≥ 95.7% to ciprofloxacin, imipenem, meropenem, piperacillin, ceftazidime, cefepime, cefoperazone/sulbactam, amikacin, tobramycin, and gentamicin; however, they exhibited a susceptibility of only 69.8% to aztreonam. They exhibited substantial resistances to ceftriaxone, enrofloxacin, cefotaxime, and moxalactam. S. marcescens isolates exhibited high susceptibilities of ≥ 90.9% to kanamycin, ceftiofur, sulfamethoxazole-trimethoprim, and the 15 aforementioned drugs, but exhibited resistance to minocycline. Neither MBL-producers nor MDRP strains were detected among the 171 strains. The dominant serotypes of P. aeruginosa isolates were OG, OA, OB, OI, OF, OE, and OK; those of S. marcescens isolates were O6 and O5. Every S. marcescens isolate was pigmented. These findings suggest that bovine P. aeruginosa and S. marcescens isolates differ from human isolates from both antibiogram and phenotypic perspectives, and could help to evaluate differences in bacteriological characteristics between bovine and human isolates. PMID:21783330

  19. Matrix Isolation and Computational Study of iso-CF2Br2: a Route to Molecular Products in CF2Br2 Photolysis

    NASA Astrophysics Data System (ADS)

    George, Lisa; Kalume, Aimable; Reid, Scott A.; El-Khoury, Patrick Z.; Tarnovsky, Alexander

    2010-06-01

    The photolysis products of dibromodifluoromethane following selected wavelength laser irradiation were characterized by matrix isolation infrared and UV/Visible spectroscopy, supported by ab initio calculations. Photolysis at wavelengths of 240 and 266 nm of CF2Br2:Ar samples (1:5000) held at 5 K yielded iso-CF2Br2 (F2CBrBr), a weakly bound isomer of CF2Br2, which is characterized here for the first time. The observed infrared and UV/Visible absorptions of iso-CF2Br2 are in excellent agreement with computational predictions at the B3LYP/aug-cc-pVTZ level. Single point energy calculations at the CCSD(T)/aug-cc-pVDZ level on the B3LYP optimized geometries show that the iso-form is a minimum on the CF2Br2 potential energy surface, lying some 55 kcal/mol above the CF2Br2 ground state. The energies of various stationary points on the CF2Br2 PES were characterized computationally; taken with our experimental results, these show that iso-CF2Br2 is an intermediate in the Br + CF2Br reaction leading to molecular products (CF2 + Br2). The photochemistry of the iso-form was also investigated; excitation into the intense 359 nm absorption band resulted in isomerization to CF2Br2. Our results are discussed in view of the rich literature on the gas-phase photochemistry of CF2Br2, particularly with respect to the existence of a roaming atom pathway leading to molecular products.

  20. Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

    PubMed Central

    Hla, S W; Hui, K P; Tan, W C; Ho, B

    1996-01-01

    The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis. PMID:8904417

  1. 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

  2. Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formation.

    PubMed

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

    2010-08-01

    Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonization by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA FISH and confocal laser scanning microscopy. Among the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect, and even after 6 h, S. epidermidis levels in dual-species biofilms were reduced by >85% compared with those without P. aeruginosa. Interestingly, strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa, which may develop over time in chronic infections, could counteract colonization by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon. PMID:20579097

  3. Molecular surveillance for carbapenemase genes in carbapenem-resistant Pseudomonas aeruginosa in Australian patients with cystic fibrosis.

    PubMed

    Tai, Anna S; Kidd, Timothy J; Whiley, David M; Ramsay, Kay A; Buckley, Cameron; Bell, Scott C

    2015-02-01

    The aim of this study was to assess the prevalence of acquired carbapenemase genes amongst carbapenem non-susceptible Pseudomonas aeruginosa isolates in Australian patients with cystic fibrosis (CF). Cross-sectional molecular surveillance for acquired carbapenemase genes was performed on CF P. aeruginosa isolates from two isolate banks comprising: (i) 662 carbapenem resistant P. aeruginosa isolates from 227 patients attending 10 geographically diverse Australian CF centres (2007-2009), and (ii) 519 P. aeruginosa isolates from a cohort of 173 adult patients attending one Queensland CF clinic in 2011. All 1189 P. aeruginosa isolates were tested by polymerase chain reaction (PCR) protocols targeting ten common carbapenemase genes, as well the Class 1 integron intI1 gene and the aadB aminoglycoside resistance gene. No carbapenemase genes were identified among all isolates tested. The intI1 and aadB genes were frequently detected and were significantly associated with the AUST-02 strain (OR 24.6, 95% CI 9.3-65.6; p < 0.0001) predominantly from Queensland patients. Despite the high prevalence of carbapenem resistance in P. aeruginosa in Australian patients with CF, no acquired carbapenemase genes were detected in the study, suggesting chromosomal mutations remain the key resistance mechanism in CF isolates. Systematic surveillance for carbapenemase-producing P. aeruginosa in CF by molecular surveillance is ongoing. PMID:25551306

  4. A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa

    PubMed Central

    Nair, Anusree V.; Joseph, Neetha; Krishna, Kiran; Sneha, K. G.; Tom, Neenu; Jangid, Kamlesh; Nair, Shanta

    2015-01-01

    Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria. PMID:26413053

  5. Pseudomonas aeruginosa isolates in severe chronic obstructive pulmonary disease: characterization and risk factors

    PubMed Central

    2014-01-01

    Background Patients with severe chronic obstructive pulmonary disease (COPD) are at increased risk of infection by P. aeruginosa. The specific role of bronchiectasis in both infection and chronic colonization by this microorganism in COPD, however, remains ill defined. To evaluate the prevalence and risk factors for P. aeruginosa recovery from sputum in outpatients with severe COPD, characterizing P. aeruginosa isolates by pulsed-field gel electrophoresis (PFGE) and focusing on the influence of bronchiectasis on chronic colonization in these patients. Methods A case-cohort study of 118 patients with severe COPD attended at a Respiratory Day Unit for an acute infectious exacerbation and followed up over one year. High-resolution CT scans were performed during stability for bronchiectasis assessment and sputum cultures were obtained during exacerbation and stability in all patients. P. aeruginosa isolates were genotyped by PFGE. Determinants of the recovery of P. aeruginosa in sputum and chronic colonization by this microorganism were assessed by multivariate analysis. Results P. aeruginosa was isolated from 41 of the 118 patients studied (34.7%). Five of these 41 patients (12.2%) with P. aeruginosa recovery fulfilled criteria for chronic colonization. In the multivariate analysis, the extent of bronchiectasis (OR 9.8, 95% CI: 1.7 to 54.8) and the number of antibiotic courses (OR 1.7, 95% CI: 1.1 to 2.5) were independently associated with an increased risk of P. aeruginosa isolation. Chronic colonization was unrelated to the presence of bronchiectasis (p=0.75). In patients with chronic colonization the isolates of P. aeruginosa retrieved corresponded to the same clones during the follow-up, and most of the multidrug resistant isolates (19/21) were harbored by these patients. Conclusions The main risk factors for P. aeruginosa isolation in severe COPD were the extent of bronchiectasis and exposure to antibiotics. Over 10% of these patients fulfilled criteria for

  6. Characterization of Toxin-Antitoxin (TA) Systems in Pseudomonas aeruginosa Clinical Isolates in Iran

    PubMed Central

    Savari, Mohammad; Rostami, Soodabeh; Ekrami, Alireza; Bahador, Abbas

    2016-01-01

    Background: Pseudomonas aeruginosa is among the most problematic hospital and community-acquired pathogens. Toxin-antitoxin (TA) systems are maintenance regulatory systems in bacteria and have recently been considered new targets for antimicrobial therapy. The prevalence and transcription of these systems in clinical isolates are still unknown. Objectives: The aim of this study was to characterize three types of TA systems (parDE, relBE, and higBA) among P. aeruginosa clinical isolates. Materials and Methods: We typed our clinical isolates by ERIC-PCR (enterobacterial repetitive intergenic consensus sequence-based polymerase chain reaction) and BOX-PCR. We then investigated 174 P. aeruginosa clinical isolates from three hospitals in Ahvaz, Iran, for the presence of TA system genes, and determined whether these systems were encoded on chromosomes or plasmids by amplification of the flanking regions. Results: Our results showed that in the 174 P. aeruginosa isolates, relBE and higBA were universal, but parDE was less prevalent. Both of the flanking regions of the parDE genes in all positive isolates were amplified. The flanking regions of nearly all relBE genes were amplified. Amplification was observed for the downstream sequence of every higBA locus, as well as for the region upstream of higBA, except in 14 strains. Conclusions: Based on the presence of TA systems in the majority of P. aeruginosa isolates, these could be used as a novel target for antimicrobial therapy. PMID:27099681

  7. Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts.

    PubMed

    Lorè, Nicola Ivan; Cigana, Cristina; De Fino, Ida; Riva, Camilla; Juhas, Mario; Schwager, Stephan; Eberl, Leo; Bragonzi, Alessandra

    2012-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host. PMID:22558188

  8. Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts

    PubMed Central

    De Fino, Ida; Riva, Camilla; Juhas, Mario; Schwager, Stephan; Eberl, Leo; Bragonzi, Alessandra

    2012-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host. PMID:22558188

  9. 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

  10. Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean▿

    PubMed Central

    Khan, Nurul Huda; Ahsan, Mahbuba; Yoshizawa, Susumu; Hosoya, Shoichi; Yokota, Akira; Kogure, Kazuhiro

    2008-01-01

    Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes. PMID:18757570

  11. Photodynamic antimicrobial therapy to inhibit pseudomonas aeruginosa of corneal isolates (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Durkee, Heather A.; Relhan, Nidhi; Arboleda, Alejandro; Halili, Francisco; De Freitas, Carolina; Alawa, Karam; Aguilar, Mariela C.; Amescua, Guillermo; Miller, Darlene; Parel, Jean-Marie

    2016-03-01

    Keratitis associated with Pseudomonas aeruginosa is difficult to manage. Treatment includes antibiotic eye drops, however, some strains of Pseudomonas aeruginosa are resistant. Current research efforts are focused on finding alternative and adjunct therapies to treat multi-drug resistant bacteria. One promising alternate technique is photodynamic therapy (PDT). The purpose of this study was to evaluate the effect of riboflavin- and rose bengal-mediated PDT on Pseudomonas aeruginosa keratitis isolates in vitro. Two isolates (S+U- and S-U+) of Pseudomonas aeruginosa were derived from keratitis patients and exposed to five experimental groups: (1) Control (dark, UV-A irradiation, 525nm irradiation); (2) 0.1% riboflavin (dark, UV-A irradiation); and (3) 0.1% rose bengal, (4) 0.05% rose bengal and (5) 0.01% rose bengal (dark, 525nm irradiation). Three days after treatment, in dark conditions of all concentration of riboflavin and rose bengal showed no inhibition in both S+U- and S-U+ strains of Pseudomonas aeruginosa. In 0.1% and 0.05% rose bengal irradiated groups, for both S+U- and S-U+ strains, there was complete inhibition of bacterial growth in the central 50mm zone corresponding to the diameter of the green light source. These in vitro results suggest that rose bengal photodynamic therapy may be an effective adjunct treatment for Pseudomonas aeruginosa keratitis.

  12. Genetic characterization of Microcystis aeruginosa isolates from Portuguese freshwater systems.

    PubMed

    Moreira, Cristiana; Vasconcelos, Vitor; Antunes, Agostinho

    2016-07-01

    Cyanobacteria are microorganisms that pose a serious threat to the aquatic waterways through the production of dense blooms under eutrophic conditions and the release of toxic secondary metabolites-cyanotoxins. Within cyanobacteria, the colonial planktonic Microcystis aeruginosa is widely distributed in both fresh and brackish aquatic environments throughout the world being frequently observed in the Portuguese water systems. Apart from the well-established distribution of M. aeruginosa in Portugal, knowledge of its genetic diversity and population structure is unknown. Therefore, in this study twenty-seven strains were obtained from the North, Centre and South regions of Portugal and were subjected to extensive phylogenetic analyses using simultaneously four distinct genetic markers (16S rRNA, 16S-23S ITS, DNA gyrase subunit ß and cell division protein (ftsZ)) encompassing in total 2834 bp. With this work we characterized the phylogenetic relationship among the Portuguese strains, with the southern strains showing higher genetic structure relatively to the North and Centre strains. A total of fifteen genotypes were determined for M. aeruginosa in Portuguese water systems revealing a high genetic diversity. This is also the first study to report geographic variation on the population structure of the Portuguese M. aeruginosa. PMID:27263013

  13. Antimicrobial testing of selected fluoroquinolones against Pseudomonas aeruginosa isolated from canine otitis.

    PubMed

    McKay, Lindsay; Rose, Crystal D Schuman; Matousek, Jennifer L; Schmeitzel, Lynn S; Gibson, Nicole M; Gaskin, Jack M

    2007-01-01

    A total of 100 Pseudomonas aeruginosa (P. aeruginosa) isolates were collected over a 1.5-year period from cases of canine otitis. Sensitivities to enrofloxacin, marbofloxacin, and orbifloxacin were determined using minimum inhibitory concentration testing (MICT). Isolates were also tested for sensitivities to enrofloxacin and marbofloxacin using disk-diffusion susceptibility testing (DDST). Isolates were significantly more sensitive to marbofloxacin than to enrofloxacin (z = -4.57; P<0.05) or orbifloxacin (z = -5.02; P<0.05). Agreement was 87% between MICT and DDST for marbofloxacin, with approximately equal numbers of overestimation and underestimation errors. Agreement was 74% between MICT and DDST for enrofloxacin, but DDST tended to overestimate the number of enrofloxacin-susceptible strains. These results suggest that marbofloxacin is more effective against P. aeruginosa than either enrofloxacin or orbifloxacin and that relying on DDST may lead to ineffective enrofloxacin treatment. PMID:17975212

  14. Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network

    PubMed Central

    Slekovec, Céline; Plantin, Julie; Cholley, Pascal; Thouverez, Michelle; Talon, Daniel; Bertrand, Xavier; Hocquet, Didier

    2012-01-01

    The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×106 CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination. PMID:23284623

  15. Antimicrobial resistance and molecular typing of pseudomonas aeruginosa isolated from surgical wounds in Lagos, Nigeria.

    PubMed

    Smith, Stella; Ganiyu, Olaniyi; John, Rachael; Fowora, Muinah; Akinsinde, Kehinde; Odeigah, Peter

    2012-01-01

    The aim of the study was to determine the resistance patterns of Pseudomonas aeruginosa isolates recovered from patients with surgical wounds in hospitals and also to investigate their epidemiological relatedness using molecular typing techniques. Twenty Pseudomonas sp. isolated from surgical wounds were subjected to antibiotic susceptibility testing by disk diffusion, plasmid profile, SDS-PAGE and PCR using the parC, gyr A gene and RAPD using the 1254 primer. The isolates showed resistance to 12 different antibiotics with six being 100% resistant. Plasmids were detected in 16 (80%) of the isolates. The RAPD-PCR using the primer 1254, SDS-PAGE classified the 20 Pseudomonas spp. into 5 and 6 types respectively. Pseudomona aeruginosa strains isolated from surgical wounds were generally resistant to a broad range of antibiotics and this is rather worrisome. The typing techniques classified the 20 isolates into 5 and 6 groups. PMID:22837123

  16. Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa.

    PubMed

    Daniels, Jonathan B; Scoffield, Jessica; Woolnough, Jessica L; Silo-Suh, Laura

    2014-12-01

    Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits. PMID:25409940

  17. Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. Results In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. Conclusions Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed. PMID:24773920

  18. Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

    PubMed Central

    Cifani, Noemi; Pompili, Barbara; Anile, Marco; Patella, Miriam; Diso, Daniele; Venuta, Federico; Cimino, Giuseppe; Quattrucci, Serena; Di Domenico, Enea Gino; Ascenzioni, Fiorentina; Porto, Paola Del

    2013-01-01

    Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations. PMID:23977124

  19. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia

    PubMed Central

    Jeannot, Katy; El-Mahdy, Taghrid S.; Samaha, Hassan A.; Shibl, Atef M.; Plésiat, Patrick; Courvalin, Patrice

    2016-01-01

    Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia. PMID:27597874

  20. Diversity of Molecular Mechanisms Conferring Carbapenem Resistance to Pseudomonas aeruginosa Isolates from Saudi Arabia.

    PubMed

    Al-Agamy, Mohamed H; Jeannot, Katy; El-Mahdy, Taghrid S; Samaha, Hassan A; Shibl, Atef M; Plésiat, Patrick; Courvalin, Patrice

    2016-01-01

    Background. This study described various molecular and epidemiological characters determining antibiotic resistance patterns in Pseudomonas aeruginosa isolates. Methods. A total of 34 carbapenem-resistant P. aeruginosa clinical isolates were isolated from samples collected at a tertiary hospital in Riyadh, Saudi Arabia, from January to December 2011. Susceptibility testing, serotyping, molecular characterization of carbapenem resistance, and pulsed-field gel electrophoresis (PFGE) were performed. Results. All isolates were resistant to ceftazidime, and more than half were highly resistant (minimum inhibitory concentration (MIC) > 256 mg/L). Fifteen isolates had MIC values ≥64 mg/L for any of the carbapenems examined. Vietnamese extended-spectrum β-lactamase (VEB-1) (n = 16/34) and oxacillinase (OXA-10) (n = 14/34) were the most prevalent extended-spectrum β-lactamase and penicillinase, respectively. Verona imipenemase (VIM-1, VIM-2, VIM-4, VIM-11, and VIM-28) and imipenemase (IMP-7) variants were found in metallo-β-lactamase producers. A decrease in outer membrane porin gene (oprD) expression was seen in nine isolates, and an increase in efflux pump gene (MexAB) expression was detected in five isolates. Six serotypes (O:1, O:4, O:7, O:10, O:11, and O:15) were found among the 34 isolates. The predominant serotype was O:11 (16 isolates), followed by O:15 (nine isolates). PFGE analysis of the 34 carbapenem-resistant P. aeruginosa isolates revealed 14 different pulsotypes. Conclusions. These results revealed diverse mechanisms conferring carbapenem resistance to P. aeruginosa isolates from Saudi Arabia. PMID:27597874

  1. Effect of Tyrosol and Farnesol on Virulence and Antibiotic Resistance of Clinical Isolates of Pseudomonas aeruginosa

    PubMed Central

    Hassan Abdel-Rhman, Shaymaa; Mostafa El-Mahdy, Areej; El-Mowafy, Mohammed

    2015-01-01

    Mixed-species biofilms could create a protected environment that allows for survival to external antimicrobials and allows different bacterial-fungal interactions. Pseudomonas aeruginosa-Candida albicans coexistence is an example for such mixed-species community. Numerous reports demonstrated how P. aeruginosa or its metabolites could influence the growth, morphogenesis, and virulence of C. albicans. In this study, we investigated how the C. albicans quorum sensing compounds, tyrosol and farnesol, might affect Egyptian clinical isolates of P. aeruginosa regarding growth, antibiotic sensitivity, and virulence. We could demonstrate that tyrosol possesses an antibacterial activity against P. aeruginosa (10 µM inhibited more than 50% of growth after 16 h cultivation). Moreover, we could show for the first time that tyrosol strongly inhibits the production of the virulence factors hemolysin and protease in P. aeruginosa, whereas farnesol inhibits, to lower extent, hemolysin production in this bacterial pathogen. Cumulatively, tyrosol is expected to strongly affect P. aeruginosa in mixed microbial biofilm. PMID:26844228

  2. Cystic Fibrosis Isolates of Pseudomonas aeruginosa Retain Iron-Regulated Antimicrobial Activity against Staphylococcus aureus through the Action of Multiple Alkylquinolones.

    PubMed

    Nguyen, Angela T; Jones, Jace W; Cámara, Miguel; Williams, Paul; Kane, Maureen A; Oglesby-Sherrouse, Amanda G

    2016-01-01

    Cystic fibrosis (CF) is a hereditary disease that predisposes individuals to pulmonary dysfunction and chronic infections. Early infection of the CF lung with Staphylococcus aureus is common, while Pseudomonas aeruginosa becomes dominant as disease progresses. Emergence of P. aeruginosa likely depends on the action of multiple 2-alkyl-4-(1H)-quinolones (AQ) secreted by this organism. We recently showed that antimicrobial activity against S. aureus is enhanced by iron depletion and is dependent upon multiple AQ metabolites. Two of these AQs, the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone] and 2-heptyl-4-hydroxyquinoline (HHQ), are quorum sensing molecules that activate the expression of multiple microbicidal factors. Here we show for the first time that HHQ also exhibits innate antimicrobial activity against S. aureus. We further show that iron depletion potentiates the antistaphylococcal activity of HHQ, as well as 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), another AQ that functions as a cytochrome B inhibitor. Notably, we found that deletion of the genes for the terminal biosynthetic steps for either PQS or HQNO results in overproduction of the HHQ intermediate, likely maintaining the ability of these mutants to mediate antimicrobial activity. Compensatory increases in HHQ were also observed in PQS-deficient CF isolates, which also retained the ability to mediate iron-regulated antimicrobial activity against S. aureus. These studies demonstrate that iron-regulated antimicrobial activity of P. aeruginosa against S. aureus is due to the cumulative effects of multiple AQ metabolites, both the production and activity of which are modulated by environmental iron levels. PMID:27512392

  3. Cystic Fibrosis Isolates of Pseudomonas aeruginosa Retain Iron-Regulated Antimicrobial Activity against Staphylococcus aureus through the Action of Multiple Alkylquinolones

    PubMed Central

    Nguyen, Angela T.; Jones, Jace W.; Cámara, Miguel; Williams, Paul; Kane, Maureen A.; Oglesby-Sherrouse, Amanda G.

    2016-01-01

    Cystic fibrosis (CF) is a hereditary disease that predisposes individuals to pulmonary dysfunction and chronic infections. Early infection of the CF lung with Staphylococcus aureus is common, while Pseudomonas aeruginosa becomes dominant as disease progresses. Emergence of P. aeruginosa likely depends on the action of multiple 2-alkyl-4-(1H)-quinolones (AQ) secreted by this organism. We recently showed that antimicrobial activity against S. aureus is enhanced by iron depletion and is dependent upon multiple AQ metabolites. Two of these AQs, the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone] and 2-heptyl-4-hydroxyquinoline (HHQ), are quorum sensing molecules that activate the expression of multiple microbicidal factors. Here we show for the first time that HHQ also exhibits innate antimicrobial activity against S. aureus. We further show that iron depletion potentiates the antistaphylococcal activity of HHQ, as well as 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), another AQ that functions as a cytochrome B inhibitor. Notably, we found that deletion of the genes for the terminal biosynthetic steps for either PQS or HQNO results in overproduction of the HHQ intermediate, likely maintaining the ability of these mutants to mediate antimicrobial activity. Compensatory increases in HHQ were also observed in PQS-deficient CF isolates, which also retained the ability to mediate iron-regulated antimicrobial activity against S. aureus. These studies demonstrate that iron-regulated antimicrobial activity of P. aeruginosa against S. aureus is due to the cumulative effects of multiple AQ metabolites, both the production and activity of which are modulated by environmental iron levels. PMID:27512392

  4. Emergence of Carbapenem-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Clinical Isolates Collected from Some Libyan Hospitals.

    PubMed

    Mathlouthi, Najla; Areig, Zaynab; Al Bayssari, Charbel; Bakour, Sofiane; Ali El Salabi, Allaaeddin; Ben Gwierif, Salha; Zorgani, Abdulaziz A; Ben Slama, Karim; Chouchani, Chedly; Rolain, Jean-Marc

    2015-06-01

    The aim of the present study was to investigate the molecular mechanism of carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates recovered from Libyan hospitals between April 2013 and April 2014. In total, 49 strains (24 P. aeruginosa and 25 A. baumannii) were isolated, including 21 P. aeruginosa and 22 A. baumannii isolates (87.75%) resistant to imipenem (minimum inhibitory concentrations ≥16 μg/ml). The blaVIM-2 gene was detected in 19 P. aeruginosa isolates. All imipenem-resistant P. aeruginosa isolates showed the presence of OprD mutations. Acquired OXA-carbapenemase-encoding genes were present in all A. baumannii isolates: blaOXA-23 (n=19) and blaOXA-24 (n=3). Finally, a total of 13 and 17 different sequence types were assigned to the 21 P. aeruginosa and the 22 A. baumannii carbapenem-resistant isolates, respectively. This study is the first report describing imipenem-resistant P. aeruginosa and A. baumannii isolated from patients in Libya. We report the first case of co-occurrence of blaVIM-2 with oprD porin loss in identical isolates of P. aeruginosa in Libya and demonstrate that these oprD mutations can be used as a tool to study the clonality in P. aeruginosa isolates. We also report the first identification of multidrug-resistant A. baumannii isolates harboring blaOXA-23-like, blaOXA-24-like, and blaOXA-48-like genes in Libya. PMID:25587875

  5. The identification, typing, and antimicrobial susceptibility of Pseudomonas aeruginosa isolated from mink with hemorrhagic pneumonia.

    PubMed

    Qi, Jing; Li, Lulu; Du, Yijun; Wang, Shourong; Wang, Jinwen; Luo, Yanbo; Che, Jie; Lu, Jinxing; Liu, Hui; Hu, Guangchun; Li, Jixia; Gong, Yanwen; Wang, Guisheng; Hu, Ming; Shiganyan; Liu, Yuqing

    2014-06-01

    The biological characteristics and molecular epidemiology of Pseudomonas aeruginosa associated with mink hemorrhagic pneumonia from Shandong province of eastern China were determined in this study. From 2010 to 2011, 30 mink P. aeruginosa isolates were identified from lung, fecal and feed samples of clinical cases and subjected to serotyping, antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) using SpeI. The P. aeruginosa isolates belonged to four serotypes-21 of type G, four of type I, three of type M, one of type B, and one non-typable strain. The strains were divided into four large groups as determined by PFGE. Isolates from the group 2 were highly homologous and were obtained from the same region as an epidemic. All of the isolates were sensitive to piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, amikacin, gentamicin and tobramycin and resistant to ampicillin, cefuroxime and cefuroxime axetil. A high frequency of resistance was found to ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, nitrofurantoin, and trimethoprim/sulfamethoxazole (96.7%). Resistance to ticarcillin/clavulanic acid, ciprofloxacin and levofloxacin was less common (13.3%). There was no relationship between antibiotic resistance and serotype distribution of the isolates. The epidemic serotype of P. aeruginosa from the mink hemorrhagic pneumonia in Shandong province was type G, which was a clone of commonly found in this province. These findings reveal the genetic similarities and antimicrobial susceptibility profiles of P. aeruginosa from clinical cases of mink hemorrhagic pneumonia and will facilitate the prevention and control of the disease in Shandong province of China. PMID:24629901

  6. Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment-detected in-hospital transmission.

    PubMed

    Johansson, Ewa; Welinder-Olsson, Christina; Gilljam, Marita

    2014-02-01

    Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. PMID:24450429

  7. 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

  8. Bacteriophage can lyse antibiotic-resistant Pseudomonas aeruginosa isolated from canine diseases

    PubMed Central

    FURUSAWA, Takaaki; IWANO, Hidetomo; HIGUCHI, Hidetoshi; YOKOTA, Hiroshi; USUI, Masaru; IWASAKI, Tomohito; TAMURA, Yutaka

    2016-01-01

    Pseudomonas aeruginosa is a pathogen frequently identified as the cause of diverse infections or chronic disease. This microbe has natural resistance to several kinds of antibiotics, because of the species’ outer membrane, efflux pumps and growth as a biofilm. This bacterium can acquire increased resistance with specific point mutations. Bacteriophage (phage), however, can lyse these bacteria. Therefore, in the present study, we assessed the host range of phages isolates and their ability to lyse antibiotic-resistant P. aeruginosa. Present phages could lyse many strains of P. aeruginosa (28/39), including strains with high resistance to fluoroquinolones (4/6). In conclusion, application of phages for antibiotic-resistant bacteria is greatly effective. To avoid pervasive antibiotic-resistant bacteria, further development of phage usage for disease treatment is required. PMID:26876365

  9. Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates

    PubMed Central

    Iebba, Valerio; Totino, Valentina; Santangelo, Floriana; Gagliardi, Antonella; Ciotoli, Luana; Virga, Alessandra; Ambrosi, Cecilia; Pompili, Monica; De Biase, Riccardo V.; Selan, Laura; Artini, Marco; Pantanella, Fabrizio; Mura, Francesco; Passariello, Claudio; Nicoletti, Mauro; Nencioni, Lucia; Trancassini, Maria; Quattrucci, Serena; Schippa, Serena

    2014-01-01

    Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) “static” biofilms; (3) field emission scanning electron microscope (FESEM); (4) “flow” biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new “epibiotic” foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while “static” and “flow” S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a “living antibiotic” in CF, even if further studies are required to simulate its in vivo predatory behavior. PMID:24926292

  10. Antibiotic resistance profiles and quorum sensing-dependent virulence factors in clinical isolates of pseudomonas aeruginosa.

    PubMed

    Wang, Huafu; Tu, Faping; Gui, Zhihong; Lu, Xianghong; Chu, Weihua

    2013-06-01

    Pseudomonas aeruginosa produces multiple virulence factors that have been associated with quorum sensing. The aim of this study was to evaluate the prevalence of drug resistant profiles and quorum sensing related virulence factors. Pseudomonas aeruginosa were collected from different patients hospitalized in China, the isolates were tested for their susceptibility to different common antimicrobial drugs and detected QS-related virulence factors. We identified 170 isolates displaying impaired phenotypic activity, approximately 80 % of the isolates were found to exhibit the QS-dependent phenotypes, among them, 12 isolates were defective in AHLs production, and therefore considered QS-deficient strains. Resistance was most often observed to Cefazolin (81.2 %), followed by trimethoprim-sulfamethoxazole (73.5 %), ceftriaxone (62.4 %) and Cefotaxime, Levofloxacin, Ciprofloxacin (58.8 %), and to a lesser extent Meropenem (20.0 %), Cefepime (18.8 %), and Cefoperazone/sulbactam (2.4 %) The QS-deficient isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials. The results showed a high incidences of antibiotic resistance and virulence properties in P. aeruginosa, and indicate that the clinical use of QS-inhibitory drugs that appear superior to conventional antimicrobials by not exerting any selective pressure on resistant strains. PMID:24426103

  11. 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

  12. 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

  13. Bacterial evolution in PCD and CF patients follows the same mutational steps.

    PubMed

    Sommer, Lea M; Alanin, Mikkel Christian; Marvig, Rasmus L; Nielsen, Kim Gjerum; Høiby, Niels; von Buchwald, Christian; Molin, Søren; Johansen, Helle Krogh

    2016-01-01

    Infections with Pseudomonas aeruginosa increase morbidity in primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) patients. Both diseases are associated with a defect of the mucociliary clearance; in PCD caused by non-functional cilia, in CF by changed mucus. Whole genome sequencing of P. aeruginosa isolates from CF patients has shown that persistence of clonal lineages in the airways is facilitated by genetic adaptation. It is unknown whether this also applies to P. aeruginosa airway infections in PCD. We compared within-host evolution of P. aeruginosa in PCD and CF patients. P. aeruginosa isolates from 12 PCD patients were whole genome sequenced and phenotypically characterised. Ten out of 12 PCD patients were infected with persisting clone types. We identified convergent evolution in eight genes, which are also important for persistent infections in CF airways: genes related to antibiotic resistance, quorum sensing, motility, type III secretion and mucoidity. We document phenotypic and genotypic parallelism in the evolution of P. aeruginosa across infected patients with different genetic disorders. The parallel changes and convergent adaptation and evolution may be caused by similar selective forces such as the intensive antibiotic treatment and the inflammatory response, which drive the evolutionary processes. PMID:27349973

  14. Bacterial evolution in PCD and CF patients follows the same mutational steps

    PubMed Central

    Sommer, Lea M.; Alanin, Mikkel Christian; Marvig, Rasmus L.; Nielsen, Kim Gjerum; Høiby, Niels; von Buchwald, Christian; Molin, Søren; Johansen, Helle Krogh

    2016-01-01

    Infections with Pseudomonas aeruginosa increase morbidity in primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) patients. Both diseases are associated with a defect of the mucociliary clearance; in PCD caused by non-functional cilia, in CF by changed mucus. Whole genome sequencing of P. aeruginosa isolates from CF patients has shown that persistence of clonal lineages in the airways is facilitated by genetic adaptation. It is unknown whether this also applies to P. aeruginosa airway infections in PCD. We compared within-host evolution of P. aeruginosa in PCD and CF patients. P. aeruginosa isolates from 12 PCD patients were whole genome sequenced and phenotypically characterised. Ten out of 12 PCD patients were infected with persisting clone types. We identified convergent evolution in eight genes, which are also important for persistent infections in CF airways: genes related to antibiotic resistance, quorum sensing, motility, type III secretion and mucoidity. We document phenotypic and genotypic parallelism in the evolution of P. aeruginosa across infected patients with different genetic disorders. The parallel changes and convergent adaptation and evolution may be caused by similar selective forces such as the intensive antibiotic treatment and the inflammatory response, which drive the evolutionary processes. PMID:27349973

  15. Prevalence of ESBLs-producing Pseudomonas aeruginosa isolates from different wards in a Chinese teaching hospital

    PubMed Central

    Chen, Zhilong; Niu, Hui; Chen, Guangyu; Li, Mingcheng; Li, Ming; Zhou, Yuqing

    2015-01-01

    This study was to explore the molecular dissemination of P. aeruginosa producing extended spectrum β-lactamase (ESBLs) recovered from the different wards in a teaching hospital, Jilin. Among 240 isolates, 91 strains were isolated from burn wards and 149 strains from surgical wards. A total of 210 strains (87.5%) produced ESBLs, 30 strains (12.5%) didn’t produce ESBLs. All ESBLs isolates showed identical antimicrobial susceptibility profiles. The genotypic prevalence of ESBLs for bla SHV-12, bla TEM-24, bla CTX-M-1, bla CTX-M-2, bla CTX-M-3, bla PER and bla VEB genes was 17.6%, 20.5%, 14.3%, 9.6%, 12.9%, 13.8% and 11.4% respectively. All P. aeruginosa strains producing ESBLs had three to six plasmids and contained class 1 integrons, which transferred resistance to E. coli C 600 by conjuation. The data indicated a high prevalence of ESBL among P. aeruginosa isolates in this region and their enzyme types were diverse. PMID:26770582

  16. Genome Analysis of Environmental and Clinical P. aeruginosa Isolates from Sequence Type-1146

    PubMed Central

    Sánchez, David; Gomila, Margarita; Bennasar, Antonio; Lalucat, Jorge; García-Valdés, Elena

    2014-01-01

    The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies. PMID:25329302

  17. Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates.

    PubMed

    Scaccabarozzi, Licia; Leoni, Livia; Ballarini, Annalisa; Barberio, Antonio; Locatelli, Clara; Casula, Antonio; Bronzo, Valerio; Pisoni, Giuliano; Jousson, Olivier; Morandi, Stefano; Rapetti, Luca; García-Fernández, Aurora; Moroni, Paolo

    2015-01-01

    Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates. PMID:26606430

  18. Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

    PubMed Central

    Scaccabarozzi, Licia; Leoni, Livia; Ballarini, Annalisa; Barberio, Antonio; Locatelli, Clara; Casula, Antonio; Bronzo, Valerio; Pisoni, Giuliano; Jousson, Olivier; Morandi, Stefano; Rapetti, Luca; García-Fernández, Aurora; Moroni, Paolo

    2015-01-01

    Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates. PMID:26606430

  19. Resistance of Pseudomonas aeruginosa Isolates to Hydrogel Contact Lens Disinfection Correlates with Cytotoxic Activity

    PubMed Central

    Lakkis, Carol; Fleiszig, Suzanne M. J.

    2001-01-01

    One of the most common pathogens in infection of hydrogel contact lens wearers is Pseudomonas aeruginosa, which can gain access to the eye via contamination of the lens, lens case, and lens care solutions. Only one strain per species is used in current regulatory testing for the marketing of chemical contact lens disinfectants. The aim of this study was to determine whether P. aeruginosa strains vary in their susceptibility to hydrogel contact lens disinfectants. A method for rapidly screening bacterial susceptibility to contact lens disinfectants was developed, based on measurement of the MIC. The susceptibility of 35 P. aeruginosa isolates to two chemical disinfectants was found to vary among strains. MICs ranged from 6.25 to 100% for both disinfectants at 37°C, and a number of strains were not inhibited by a 100% disinfectant concentration in the lens case environment at room temperature (22°C). Resistance to disinfection appeared to be an inherent rather than acquired trait, since some resistant strains had been isolated prior to the introduction of the disinfectants and some susceptible P. aeruginosa strains could not be made more resistant by repeated disinfectant exposure. A number of P. aeruginosa strains which were comparatively more resistant to short-term disinfectant exposure also demonstrated the ability to grow to levels above the initial inoculum in one chemical disinfectant after long-term (24 to 48 h) disinfectant exposure. Resistance was correlated with acute cytotoxic activity toward corneal epithelial cells and with exsA, which encodes a protein that regulates cytotoxicity via a complex type III secretion system. These results suggest that chemical disinfection solutions may select for contamination with cytotoxic strains. Further investigation of the mechanisms and factors responsible for resistance may also lead to strategies for reducing adverse responses to contact lens wear. PMID:11283074

  20. Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

    PubMed Central

    Wang, J; Mushegian, A; Lory, S; Jin, S

    1996-01-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa. Images Fig. 2 Fig. 4 PMID:8816818

  1. 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

  2. Diverse Mobilized Class 1 Integrons Are Common in the Chromosomes of Pathogenic Pseudomonas aeruginosa Clinical Isolates

    PubMed Central

    Martinez, Elena; Marquez, Carolina; Ingold, Ana; Merlino, John; Djordjevic, Steven P.; Roy Chowdhury, Piklu

    2012-01-01

    Eleven clinical class 1 integron-containing Pseudomonas aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands. These elements seem to be undergoing successful dispersal by lateral gene transfer since integrons were identified across several lineages and more than one clonal line. PMID:22271862

  3. Prevalence and Susceptibility Pattern of Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa in Karachi

    PubMed Central

    Khan, Fouzia; Khan, Adnan; Kazmi, Shahana Urooj

    2014-01-01

    Objective: To determine the frequency and susceptibility pattern of multi-drug resistant (MDR) Pseudomonas aeruginosa isolated from clinical specimens in Karachi. Methods: This cross sectional study was conducted in Microbiology Department, University of Karachi, from January 2012 to January 2013. Clinical specimens were collected from different hospitals of Karachi. Clinical isolates were identified by standard and specific microbiological methods. The antibiotic susceptibility pattern was determined by Kirby Bauer Disc diffusion method. Clinical and Laboratory Standards Institute (CLSI) guidelines were used to determine the results. Results: The frequency of MDR P. aeruginosa isolated from different clinical specimens was found to be 30%. Amikacin was found to be the most effective antibiotic, followed by Co-trimaxazole and Quinolones. Conclusion: Antibiotic resistant P. aeruginosa are emerging as a critical human health issue. There is an urgent need to resolve the issue by taking some preventive measures. Combined efforts of health care professionals and researchers are required to educate people about the proper use of antibiotics and other infection control measures. PMID:25225505

  4. Isolation of a Poterioochromonas capable of feeding on Microcystis aeruginosa and degrading microcystin-LR.

    PubMed

    Zhang, Xue; Hu, Hong-Ying; Hong, Yu; Yang, Jia

    2008-11-01

    Algal blooms have become a worldwide issue recently, especially those comprised of toxic cyanobacteria. Grazers' predation of bloom-forming algae plays an important role in water clearing. In this study, a species of golden alga (strain ZX1), capable of feeding on the toxic cyanobacteria Microcystis aeruginosa, was isolated and identified as Poterioochromonas sp. (GenBank accession: EU586184) on the basis of morphological characteristics and 18s rRNA gene sequencing. Feeding experiments showed that ZX1 could clear high densities of M. aeruginosa (7.3 x 10(5)-4.3 x 10(6) cells mL(-1)) in a short time (40 h), with inhibition ratios higher than 99.9%. ZX1 grew during the feeding processes and achieved a maximum density of 10-20% of the initial M. aeruginosa density. Furthermore, this study is the first to report that ZX1 was able to degrade microcystin-LR (MC-LR) in cells of M. aeruginosa while digesting the whole cells, and that the degradation process was determined to be carried out inside the ZX1 cell. For a total MC-LR (intra- and extracellular) concentration of up to 114 microg L(-1), 82.7% was removed in 40 h. This study sheds light on the importance of golden alga in aquatic microbial ecosystems and in the natural transportation/transformation of MC-LR. PMID:18811657

  5. 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

  6. Successful implementation of infection control strategies prevents P. aeruginosa transmission among cystic fibrosis patients inside the hospital

    PubMed Central

    Matt, Benedikt; Mitteregger, Dieter; Renner, Sabine; Presterl, Elisabeth; Assadian, Ojan; Diab-Elschahawi, Magda

    2014-01-01

    Background: The aim of this study was to characterise the epidemiology of P. aeruginosa isolated from cystic fibrosis (CF) patients at the Vienna General Hospital (VGH) by molecular genetic fingerprinting in order to understand transmission ways and to evaluate the established infection control protocols. Methods: The outpatient clinic for CF patients at the VGH cares for children and adolescents up to the age of 18 years. Among an average of 139 patients cared for at the clinic, 41 were tested positive for P. aeruginosa during the study period. Fifty P. aeruginosa isolates, obtained between August 2010 and March 2012 from routine examinations of CF patients, were subject to molecular characterization using the DiversiLab® method. Results: 42 distinguishable molecular-biological patterns were identified, 7 of which were found multiple times. 40 out of 42 genotypes were retrieved from single patients only, while two patterns were present in two patients each. Nine patients presented with two or more phenotypically diverse P. aeruginosa isolates. In five of these cases the retrieved isolates belonged to the same genotype. Conclusion: The broad genetic heterogeneity of P. aeruginosa in the studied patient population suggests that the majority of CF patients cared for at the VGH acquire P. aeruginosa from environmental sources. It may be concluded that implemented infection control guidelines have been successful in preventing nosocomial transmission of P. aeruginosa among CF patients within the VGH and patient-to-patient transmission outside the hospital. Chronic polyclonal infection/colonization was rare in the study population. PMID:25285264

  7. Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

    PubMed Central

    Nguyen, Angela T.; O'Neill, Maura J.; Watts, Annabelle M.; Robson, Cynthia L.; Lamont, Iain L.; Wilks, Angela

    2014-01-01

    Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment. PMID:24727222

  8. Adaptation of iron homeostasis pathways by a Pseudomonas aeruginosa pyoverdine mutant in the cystic fibrosis lung.

    PubMed

    Nguyen, Angela T; O'Neill, Maura J; Watts, Annabelle M; Robson, Cynthia L; Lamont, Iain L; Wilks, Angela; Oglesby-Sherrouse, Amanda G

    2014-06-01

    Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment. PMID:24727222

  9. Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa

    PubMed Central

    Heydari, Samira; Eftekhar, Fereshteh

    2015-01-01

    Background: Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster. Objectives: The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates. Materials and Methods: Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR. Results: Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm. Conclusions: Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC

  10. Virulence Gene Profiles of Multidrug-Resistant Pseudomonas aeruginosa Isolated From Iranian Hospital Infections

    PubMed Central

    Fazeli, Nastaran; Momtaz, Hassan

    2014-01-01

    Background: The most common hospital-acquired pathogen is Pseudomonas aeruginosa. It is a multidrug resistant bacterium causing systemic infections. Objectives: The present study was carried out in order to investigate the distribution of virulence factors and antibiotic resistance properties of Pseudomonas aeruginosa isolated from various types of hospital infections in Iran. Patients and Methods: Two-hundred and seventeen human infection specimens were collected from Baqiyatallah and Payambaran hospitals in Tehran, Iran. The clinical samples were cultured immediately and samples positive for P. aeruginosa were analyzed for the presence of antibiotic resistance and bacterial virulence genes using PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed using disk diffusion methodology with Müeller–Hinton agar. Results: Fifty-eight out of 127 (45.66%) male infection specimens and 44 out of 90 (48.88%) female infection specimens harbored P. aeruginosa. Also, 65% (in male specimens) and 21% (in female specimens) of respiratory system infections were positive for P. aeruginosa, which was a high rate. The genes encoding exoenzyme S (67.64%) and phospholipases C (45.09%) were the most common virulence genes found among the strains. The incidences of various β-lactams encoding genes, including blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, and blaVEB were 94.11%, 16.66%, 15.68%, 18.62%, 21.56%, and 17.64%, respectively. The most commonly detected fluoroquinolones encoding gene was gyrA (15. 68%). High resistance levels to penicillin (100%), tetracycline (90.19%), streptomycin (64.70%), and erythromycin (43.13%) were observed too. Conclusions: Our findings should raise awareness about antibiotic resistance in hospitalized patients in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of human infections. PMID:25763199

  11. Antimicrobial susceptibility of clinical isolates of Pseudomonas aeruginosa from a Malaysian Hospital.

    PubMed

    Pathmanathan, Siva Gowri; Samat, Nor Azura; Mohamed, Ramelah

    2009-04-01

    Ongoing surveillance of Pseudomonas aeruginosa resistance against antimicrobial agents is fundamental to monitor trends in susceptibility patterns and to appropriately guide clinicians in choosing empirical or directed therapy. The in vitro activity level of eight antimicrobial drugs was assessed against 97 clinical isolates of P. aeruginosa collected consecutively for three months in 2007 from a Malaysian hospital. Antimicrobial susceptibility was determined using the E-test method in addition to the hospital's routine diagnostic testing by the disk diffusion method. Respiratory and wound swab isolates were the most frequently encountered isolates. The E-test and disk diffusion methods showed high concordance in determining the in vitro activity of the antimicrobial agents against the E isolates. Piperacillin-tazobactam was the most active antimicrobial agent with 91.8% susceptibility, followed by the aminoglycosides (amikacin, 86.6% and gentamicin, 84.5%), the quinolone (ciprofloxacin, 83.5%) and the beta-lactams (cefepime, 80.4%, ceftazidime, 80.4%, imipenem, 79.4% and meropenem, 77.3%). Incidence of multidrug resistance was 19.6% (19 out of 97 isolates). Periodic antibiotic resistance surveillance is fundamental to monitor changes in susceptibility patterns in a hospital setting. PMID:22589655

  12. Phylogenetic study of metallo-β-lactamase producing multidrug resistant Pseudomonas aeruginosa isolates from burn patients.

    PubMed

    Jena, Jayanti; Debata, Nagen Kumar; Sahoo, Rajesh Kumar; Subudhi, Enketeswara

    2015-12-01

    The present study was carried out to understand the clonal relationship using enterobacteriaceae repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) among metallo-β-lactamase (MBL) producing multidrug resistant Pseudomonas aeruginosa isolates from burn victims and their susceptibility to commonly used anti-pseudomonal agents. In the present study 94 non-duplicate P. aeruginosa strains from the wound samples of burn patients were included. Identification of the isolates was done by biochemical methods and antibiotic sensitivity was done by disc diffusion method following CLSI (Clinical Laboratory Standard Institute) guidelines. By using imipenem (IPM)-EDTA disk diffusion/double disc synergy method carbapenem resistant organisms were tested for MBL. To define the clonal relationship ERIC-PCR was used. Of the 94 isolates, 18 (19.14%) were found resistant to IPM and MBL production was shown 11 (11.70%) by the IPM-EDTA disc diffusion method. From dendrogram of the ERIC-PCR profile four major clusters were obtained (A, B, C and D). Cluster B contained the majority of the isolates (6 strains 1, 4, 8, 9, 10 and 11). This study using ERIC-PCR of randomly collected isolates exhibits high genetic diversity which rules out cross contamination frequency. PMID:26188888

  13. Aloe vera Gel: Effective Therapeutic Agent against Multidrug-Resistant Pseudomonas aeruginosa Isolates Recovered from Burn Wound Infections

    PubMed Central

    Goudarzi, Mehdi; Fazeli, Maryam; Azad, Mehdi; Seyedjavadi, Sima Sadat; Mousavi, Reza

    2015-01-01

    Objective. Aloe vera is an herbal medicinal plant with biological activities, such as antimicrobial, anticancer, anti-inflammatory, and antidiabetic ones, and immunomodulatory properties. The purpose of this study was investigation of in vitro antimicrobial activity of A. vera gel against multidrug-resistant (MDR) Pseudomonas aeruginosa isolated from patients with burn wound infections. Methods. During a 6-month study, 140 clinical isolates of P. aeruginosa were collected from patients admitted to the burn wards of a hospital in Tehran, Iran. Antimicrobial susceptibility test was carried out against the pathogens using the A. vera gel and antibiotics (imipenem, gentamicin, and ciprofloxacin). Results. The antibiogram revealed that 47 (33.6%) of all isolates were MDR P. aeruginosa. The extract isolated from A. vera has antibacterial activity against all of isolates. Also, 42 (89.4%) isolates were inhibited by A. vera gel extract at minimum inhibitory concentration (MIC) ≤ 200 µg/mL. MIC value of A. vera gel for other isolates (10.6%) was 800 µg/mL. All of MDR P. aeruginosa strains were inhibited by A. vera at similar MIC50 and MIC90 200 µg/mL. Conclusion. Based on our results, A. vera gel at various concentrations can be used as an effective antibacterial agent in order to prevent wound infection caused by P. aeruginosa. PMID:26266047

  14. High level of resistance to aztreonam and ticarcillin in Pseudomonas aeruginosa isolated from soil of different crops in Brazil.

    PubMed

    Pitondo-Silva, André; Martins, Vinicius Vicente; Fernandes, Ana Flavia Tonelli; Stehling, Eliana Guedes

    2014-03-01

    Pseudomonas aeruginosa can be found in water, soil, plants and, human and animal fecal samples. It is an important nosocomial pathogenic agent characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance through some mechanisms, among them, by the acquisition of plasmids and integrons, which are mobile genetic elements. In this study, 40 isolates from Brazilian soil were analyzed for antibiotic resistance, presence of integrons and plasmidial profile. The results demonstrated that the vast majority of the isolates have shown resistance for aztreonam (92.5%, n=37) and ticarcillin (85%, n=34), four isolates presented plasmids and eight isolates possess the class 1 integron. These results demonstrated that environmental isolates of P. aeruginosa possess surprising antibiotic resistance profile to aztreonam and ticarcillin, two antimicrobial agents for clinical treatment of cystic fibrosis patients and other infections occurred by P. aeruginosa. PMID:24369293

  15. In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states.

    PubMed

    Velez Perez, Antonio L; Schmidt-Malan, Suzannah M; Kohner, Peggy C; Karau, Melissa J; Greenwood-Quaintance, Kerryl E; Patel, Robin

    2016-07-01

    Pseudomonas aeruginosa causes a variety of life-threatening infections, some of which are associated with planktonic and others with biofilm states. Herein, we tested the combination of the novel cephalosporin, ceftolozane, with the β-lactamase inhibitor, tazobactam, against planktonic and biofilm forms of 54 clinical isolates of P. aeruginosa, using cefepime as a comparator. MIC values were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum biofilm inhibitory concentration (MBIC) values were determined using biofilm-laden pegged lids incubated in antimicrobial challenge plates containing varying concentrations of ceftolozane/tazobactam. Pegged lids were then incubated in growth recovery plates containing cation-adjusted Mueller-Hinton broth to determine the minimum biofilm bactericidal concentration (MBBC). Ceftolozane/tazobactam was highly active against planktonic P. aeruginosa, with all 54 isolates studied testing susceptible (MIC ≤4/4μg/mL). On the other hand, 51/54 biofilm P. aeruginosa had MBICs ≥16/4μg/mL, and all 54 isolates had MBBCs >32μg/mL. Of the 54 isolates, 45 (83.3%) tested susceptible to cefepime, with the MIC50/MIC90 being 4/16μg/mL, respectively, and the MBIC90 and MBBC90 both being >256μg/mL. Although ceftolozane/tazobactam is a promising antimicrobial agent for the treatment of P. aeruginosa infections, it is not highly active against P. aeruginosa biofilms. PMID:27130477

  16. Heavy metal resistance and virulence profile in Pseudomonas aeruginosa isolated from Brazilian soils.

    PubMed

    Pitondo-Silva, André; Gonçalves, Guilherme Bartolomeu; Stehling, Eliana Guedes

    2016-08-01

    Pseudomonas aeruginosa is an opportunistic pathogen, which can have several virulence factors that confer on it the ability to cause severe, acute and chronic infections. Thus, the simultaneous occurrence of resistance to antibiotics and heavy metals associated with the presence of virulence genes is a potential threat to human health and environmental balance. This study aimed to investigate the resistance profile to heavy metals and the correlation of this phenotype of resistance to antimicrobials and to investigate the pathogenic potential of 46 P. aeruginosa isolates obtained from the soil of five Brazilian regions. The bacteria were evaluating for antimicrobial and heavy metal resistance, as well as the presence of plasmids and virulence genes. The isolates showed resistance to four different antibiotics and the majority (n = 44) had resistance to aztreonam or ticarcillin, furthermore, 32 isolates showed concomitant resistance to both of these antibiotics. A high prevalence of virulence genes was found, which highlights the pathogenic potential of the studied environmental isolates. Moreover, a high frequency of heavy metal resistance genes was also detected, however, the phenotypic results indicated that other genes and/or mechanisms should be related to heavy metal resistance. PMID:27197940

  17. DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa.

    PubMed Central

    Yonezawa, M; Takahata, M; Matsubara, N; Watanabe, Y; Narita, H

    1995-01-01

    The mutations in the quinolone resistance-determining region of the gyrA gene from clinical isolates of Pseudomonas aeruginosa were determined by DNA sequencing. The strains were isolated in 1989 and 1993. No mutations were detected in the clinical isolates in 1989, while five types of mutations were identified in the isolates in 1993. These mutations were as follows: group 1, a Thr residue to an Ile residue at position 83 (Thr-83-Ile); group 2, Asp-87-Asn; group 3, Thr-83-Ile and Asp-87-Gly; group 4, Thr-83-Ile and Asp-87-Asn; group 5, Thr-83-Ile and Asp-87-His. Three types of double mutations (groups 3, 4, and 5) have not been described previously. These mutations were homologous to the Ser-83-Leu, Asp-87-Asn, and Asp-87-Gly changes observed in Escherichia coli. Thus, DNA gyrase A subunit mutations are implicated in resistance to quinolones in P. aeruginosa as well as E. coli. PMID:8540700

  18. Prevalence of ESBLs genes among multidrug-resistant isolates of Pseudomonas aeruginosa isolated from patients in Tehran.

    PubMed

    Shahcheraghi, Freshteh; Nikbin, Vajiheh-Sadat; Feizabadi, Mohammad Mehdi

    2009-03-01

    Drug susceptibility testing and PCR assay were used to determine the antibiotic susceptibility patterns and prevalence of genes encoding five different extended spectrum betalactamases (ESBLs) (PER, VEB, SHV, GES, and TEM) among 600 isolates of Pseudomonas aeruginosa cultured from patients at two hospitals in Tehran. Susceptibility of isolates to 12 different antibiotics was tested using disk diffusion method. The MICs for ceftazidime and imipenem were also determined using microbroth dilution assay. Isolates showing MICs >or=16 for ceftazidime were subjected to PCR targeting bla(SHV), bla(PER), bla(GES), bla(VEB), and bla(TEM) genes that encode ESBL. The rates of resistance were as follows: tetracycline (92%), carbenicillin (62%), cefotaxime (56%), ceftriaxon (53%), piperacilin (46%), gentamicin (31%), piperacilin/tazobactam (28%), ceftazidime (25%), amikacin (23%), ciprofloxacin (19.5%), and imipenem (6%). Thirty-nine percent of isolates (n = 234) showed MICs >or=16 microg/ml for ceftazidime, and 5.45% showed MICs >or=16 microg/ml for imepenem. The imipenem-resistant isolates showed high rate of susceptibility to colistin (89%) and polymixin B (95.5%). The frequency of bla(VEB), bla(SHV), bla(PER), bla(GES), and bla(TEM) among the ESBL isolates (MIC >or=16) were 24%, 22%, 17%, 0%, and 9%, respectively. Isolates containing bla(VEB) were resistant to almost all tested antibiotics except imepenem. This is the first report on the existence of bla(VEB), and bla(PER) in Iran. Colistin and polymixin B are highly potent against the imipenem-resistant isolates of P. aeruginosa. PMID:19265477

  19. Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance.

    PubMed

    Boyle, Brian; Fernandez, Lucia; Laroche, Jerome; Kukavica-Ibrulj, Irena; Mendes, Caio M F; Hancock, Robert W; Levesque, Roger C

    2012-01-01

    Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR). PMID:22207740

  20. Phosphate limitation induces the intergeneric inhibition of Pseudomonas aeruginosa by Serratia marcescens isolated from paper machines

    PubMed Central

    Kuo, Pei-An; Kuo, Chih-Horng; Lai, Yiu-Kay; Graumann, Peter L; Tu, Jenn

    2013-01-01

    Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria–Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini-Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation. PMID:23398522

  1. Antibacterial Activity of Hibicuslide C on Multidrug-Resistant Pseudomonas aeruginosa Isolates.

    PubMed

    Lee, Heejeong; Choi, Hyemin; Lee, Je Chul; Lee, Yoo Chul; Woo, Eun-Rhan; Lee, Dong Gun

    2016-10-01

    Pseudomonas aeruginosa is a gram-negative bacterium that is frequently related to natural resistance to many drugs. In this work, the inhibition of growth against P. aeruginosa and multidrug-resistant P. aeruginosa (MDRPA) isolated from patients at Kyungpook National University was confirmed for hibicuslide C, essential oil components from Abutilon theophrasti. Hibicuslide C has antifungal activity with membrane disruption and apoptotic response against Candida albicans. However, its antibacterial activity was not reported yet. Cells treated with hibicuslide C was showed that its antipseudomonal activity is related to gDNA fragmentation and damage by TUNEL and gDNA electrophoresis. Furthermore, hibicuslide C worked synergistically with fluoroquinolones and rifampicin against MDRPA regardless of the ATP-associated mechanism. The antibiofilm activity possessed sole-resulting tissue culture plate method; besides that, the antibiofilm activity of other antibiotics was supported in particular MDRPA. The essential oil components like hibicuslide C may have antipseudomonal activity and, furthermore, increase in bacterial antibiotic susceptibility. PMID:27368232

  2. Susceptibility of Pseudomonas aeruginosa isolates to ceftazidime is unrelated to the expression of the outer membrane protein OprC.

    PubMed

    Pérez, F J; Navarro, D; Gimeno, C; García-de-Lomas, J

    1997-01-01

    Previously, it has been postulated that the porin OprC facilitates the diffusion of ceftazidime through the outer membrane of Pseudomonas aeruginosa. To further investigate this claim, the outer membrane protein (OMP) profiles of 22 ceftazidime-susceptible clinical isolates were analyzed. No correlation was found between MIC values and the level of expression of OprC. Further, OprC was either undetectable or expressed in reduced amounts in 12 isolates. In contrast, OprF and OprE were present in all isolates studied. This study suggests that OprC is dispensable for the permeation of ceftazidime through the outer membrane of P. aeruginosa. PMID:8996738

  3. Anti-Pseudomonas aeruginosa IgY antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils.

    PubMed

    Thomsen, Kim; Christophersen, Lars; Jensen, Peter Østrup; Bjarnsholt, Thomas; Moser, Claus; Høiby, Niels

    2016-07-01

    Moderation of polymorphonuclear neutrophils (PMNs) as part of a critical defense against invading pathogens may offer a promising therapeutic approach to supplement the antibiotic eradication of Pseudomonas aeruginosa infection in non-chronically infected cystic fibrosis (CF) patients. We have observed that egg yolk antibodies (IgY) harvested from White leghorn chickens that target P. aeruginosa opsonize the pathogen and enhance the PMN-mediated respiratory burst and subsequent bacterial killing in vitro. The effects on PMN phagocytic activity were observed in different Pseudomonas aeruginosa strains, including clinical isolates from non-chronically infected CF patients. Thus, oral prophylaxis with anti-Pseudomonas aeruginosa IgY may boost the innate immunity against Pseudomonas aeruginosa in the CF setting by facilitating a rapid and prompt bacterial clearance by PMNs. PMID:26901841

  4. Decolorization of Distillery Spent Wash Using Biopolymer Synthesized by Pseudomonas aeruginosa Isolated from Tannery Effluent

    PubMed Central

    David, Charles; Arivazhagan, M.; Balamurali, M. N.; Shanmugarajan, Dhivya

    2015-01-01

    A bacterial strain was isolated from tannery effluent which can tolerate high concentrations of potassium dichromate up to 1000 ppm. The isolated microorganism was identified as Pseudomonas aeruginosa by performing biochemical tests and molecular characterization. In the presence of excess of carbohydrate source, which is a physiological stress, this strain produces Polyhydroxybutyrate (PHB). This intracellular polymer, which is synthesized, is primarily a product of carbon assimilation and is employed by microorganisms as an energy storage molecule to be metabolized when other common energy sources are limitedly available. Efforts were taken to check whether the PHB has any positive effect on spent wash decolorization. When a combination of PHB and the isolated bacterial culture was added to spent wash, a maximum color removal of 92.77% was found which was comparatively higher than the color removed when the spent wash was treated individually with the PHB and Pseudomonas aeruginosa. PHB behaved as a support material for the bacteria to bind to it and thus develops biofilm, which is one of the natural physiological growth forms of microorganisms. The bacterial growth in the biofilm and the polymer together acted in synergy, adsorbing and coagulating the pollutants in the form of color pigments. PMID:26504787

  5. Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa in clinical settings.

    PubMed

    Mishra, Shweta; Upadhyay, Supriya; Sen, Malay Ranjan; Maurya, Anand Prakash; Choudhury, Debarati; Bhattacharjee, Amitabha

    2015-01-01

    New Delhi metallo β-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of blaNDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with blaNDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate's NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure. PMID:25635921

  6. Biodegradation of crude oil by Pseudomonas aeruginosa and Escherichia fergusonii isolated from the Goan coast.

    PubMed

    Pasumarthi, Rajesh; Chandrasekaran, Sivaraman; Mutnuri, Srikanth

    2013-11-15

    Petroleum hydrocarbons are major pollutants of the marine environment. Bioremediation is a promising approach for treating such contaminated environments. The present study aims at isolating naturally occurring bacteria from the coast of Goa, India and to study their hydrocarbonoclastic capacity. Pseudomonas aeruginosa and Escherichia fergusonii were isolated from a crude oil-contaminated sediment sample using diesel oil as the sole carbon source. The capability of the enriched culture to degrade crude oil was estimated using microcosm studies under saline conditions. Based on GC-MS analysis, the culture was found to degrade n-alkanes at a higher rate compared to polyaromatic hydrocarbons. It was also found that the culture degraded alkylated polyaromatic hydrocarbons much less than unalkylated ones. Alkanes ranging from C12 to C33 were highly degraded compared to n-C34. This study shows bioremediation of crude oil in saline (3% NaCl) conditions by naturally existing bacteria isolated from the marine environment. PMID:24045123

  7. Cooperative pathogenicity in cystic fibrosis: Stenotrophomonas maltophilia modulates Pseudomonas aeruginosa virulence in mixed biofilm

    PubMed Central

    Pompilio, Arianna; Crocetta, Valentina; De Nicola, Serena; Verginelli, Fabio; Fiscarelli, Ersilia; Di Bonaventura, Giovanni

    2015-01-01

    The present study was undertaken in order to understand more about the interaction occurring between S. maltophilia and P. aeruginosa, which are frequently co-isolated from CF airways. For this purpose, S. maltophilia RR7 and P. aeruginosa RR8 strains, co-isolated from the lung of a chronically infected CF patient during a pulmonary exacerbation episode, were evaluated for reciprocal effect during planktonic growth, adhesion and biofilm formation onto both polystyrene and CF bronchial cell monolayer, motility, as well as for gene expression in mixed biofilms. P. aeruginosa significantly affected S. maltophilia growth in both planktonic and biofilm cultures, due to an inhibitory activity probably requiring direct contact. Conversely, no effect was observed on P. aeruginosa by S. maltophilia. Compared with monocultures, the adhesiveness of P. aeruginosa on CFBE41o- cells was significantly reduced by S. maltophilia, which probably acts by reducing P. aeruginosa's swimming motility. An opposite trend was observed for biofilm formation, confirming the findings obtained using polystyrene. When grown in mixed biofilm with S. maltophilia, P. aeruginosa significantly over-expressed aprA, and algD—codifying for protease and alginate, respectively—while the quorum sensing related rhlR and lasI genes were down-regulated. The induced alginate expression by P. aeruginosa might be responsible for the protection of S. maltophilia against tobramycin activity we observed in mixed biofilms. Taken together, our results suggest that the existence of reciprocal interference of S. maltophilia and P. aeruginosa in CF lung is plausible. In particular, S. maltophilia might confer some selective “fitness advantage” to P. aeruginosa under the specific conditions of chronic infection or, alternatively, increase the virulence of P. aeruginosa thus leading to pulmonary exacerbation. PMID:26441885

  8. Pseudomonas aeruginosa isolates of distinct sub-genotypes exhibit similar potential of antimicrobial resistance by drugs exposure.

    PubMed

    Liu, Zhen-Hong; Xu, Yan; Duo, Li-Bo; Liu, Yu; Xu, Zhao-Zhen; Burns, Jane L; Liu, Gui-Rong; Yang, Bao-Feng; Liu, Shu-Lin

    2013-04-01

    Pseudomonas aeruginosa, a wide-spread opportunistic pathogen, often complicates clinical treatments due to its resistance to a large variety of antimicrobials, especially in immune compromised patients, occasionally leading to death. However, the resistance to antimicrobials varies greatly among the P. aeruginosa isolates, which raises a question on whether some sub-lineages of P. aeruginosa might have greater potential to develop antimicrobial resistance than others. To explore this question, we divided 160 P. aeruginosa isolates collected from cities of USA and China into distinct genotypes using I-CeuI, a special endonuclease that had previously been proven to reveal phylogenetic relationships among bacteria reliably due to the highly conserved 26-bp recognition sequence. We resolved 10 genotypes by I-CeuI analysis and further divided them into 82 sub-genotypes by endonuclease cleavage with SpeI. Eight of the 10 genotypes contained both multi-drug resistant (MDR) and less resistant isolates based on comparisons of their antimicrobial resistance profiles (ARPs). When the less resistant or susceptible isolates from different genotypes were exposed to eight individual antimicrobials, they showed similar potential to become resistant with minor exceptions. This is to our knowledge the first report to examine correlations between phylogenetic sub-lineages of P. aeruginosa and their potential to become resistant to antimicrobials. This study further alerts the importance and urgency of antimicrobial abuse control. PMID:23224438

  9. Flagellin induces myeloid-derived suppressor cells: implications for Pseudomonas aeruginosa infection in cystic fibrosis lung disease.

    PubMed

    Rieber, Nikolaus; Brand, Alina; Hector, Andreas; Graepler-Mainka, Ute; Ost, Michael; Schäfer, Iris; Wecker, Irene; Neri, Davide; Wirth, Andreas; Mays, Lauren; Zundel, Sabine; Fuchs, Jörg; Handgretinger, Rupert; Stern, Martin; Hogardt, Michael; Döring, Gerd; Riethmüller, Joachim; Kormann, Michael; Hartl, Dominik

    2013-02-01

    Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa-infected CF patient ex vivo-isolated as well as flagellin or P. aeruginosa in vitro-generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell-mediated host defense in CF and other P. aeruginosa-associated chronic lung diseases. PMID:23277486

  10. 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

  11. [The comparison of selected virulence factors in Pseudomonas aeruginosa catheter isolates].

    PubMed

    Olejnízková, Katerina; Holá, Veronika

    2012-05-01

    Healthcare quality improvement brings about an increasing number of invasive diagnostic and therapeutic procedures and thus also an increasing number of high-risk patients prone to hospital infections. Pseudomonas aeruginosa is one of the most commonly isolated nosocomial species and the treatment of the infection is often long and problematic, with frequent recurrences. The pathogenesis of Pseudomonas infection is associated with a range of virulence factors. In the present study, 93 catheter isolates of Pseudomonas aeruginosa were screened for the biofilm formation, motility and secretion of selected extracellular products. A high rate of the strains tested were producers of hemolysins, LasB elastase, and pyoverdines (> 70%). The biofilm formation was detected in 80% of isolates and formation of aerated biofilm was present in 90% of isolates with a positive correlation found between the two types of biofilm formation (p = 0.00583; gamma = 0.551). All strains showed swarming motility, 95% of strains showed swimming motility, and 75% of strains showed twitching motility. Among the virulence factors studied, only pyocyanin and pyochelin were produced by a lower proportion of isolates (< 25%). A positive correlation was seen between the production of some extracellular molecules (pyochelin and pyocyanin, pyocyanin and LasB elastase, and LasB elastase and haemolysins), between biofilm formation and formation of aerated biofilm, and between formation of aerated biofilm and pigments (pyoverdine and pyocyanin) production. On the other hand, a negative correlation was found between biofilm production and LasB elastase production and between the production of biofilm under immersion and pigments (pyoverdine and pyocyanin) production. All correlations are significant at the level p = 0.05, with the correlation coefficient gamma > 0.50. PMID:22880261

  12. Genetic Acquisition of NDM Gene Offers Sustainability among Clinical Isolates of Pseudomonas aeruginosa in Clinical Settings

    PubMed Central

    Mishra, Shweta; Upadhyay, Supriya; Sen, Malay Ranjan; Maurya, Anand Prakash; Choudhury, Debarati; Bhattacharjee, Amitabha

    2015-01-01

    New Delhi metallo β-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of blaNDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with blaNDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate’s NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure. PMID:25635921

  13. Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections.

    PubMed

    Rubin, J; Walker, R D; Blickenstaff, K; Bodeis-Jones, S; Zhao, S

    2008-09-18

    Infections with antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of this study was to determine antimicrobial susceptibility of 106 strains of Pseudomonas aeruginosa isolated from dogs with otitis and pyoderma from 2003 to 2006 in the United States. Three antimicrobial panels, including 6 classes and 32 antimicrobial agents, were used. A wide range of susceptibility patterns were noted with some isolates being resistant to between 8 and 28 (mean 16) of the antimicrobials tested. Among the beta-lactams, all isolates were resistant to ampicillin, cefoxitin, cefpodoxime, cephalothin and cefazolin followed by amoxicillin/clavulanic acid (99%), ceftiofur (97%), ceftriaxone (39%), cefotaxime (26%), and cefotaxime/clavulanic acid (20%), whereas less than 7% of isolates were resistant to ceftazidime/clavulanic acid, ceftazidime, piperacillin/tazobactam or cefepime. Two isolates were resistant to the carbapenems. Among the quinolones and fluoroquinolones, the most isolates were resistant to naladixic acid (96%), followed by orbifloxacin (52%), difloxacin (43%), enrofloxacin (31%), marbofloxacin (27%), gatifloxacin (23%), levofloxacin (21%), and ciprofloxacin (16%). Among the aminoglycosides, the most resistance was seen to kanamycin (90%), followed by streptomycin (69%), gentamicin (7%), and amikacin (3%). Of the remaining antimicrobials 100% of the isolates were resistant to chloramphenicol followed by tetracycline (98%), trimethoprim/sulfamethoxazole (57%), and sulfisoxazole (51%). Point mutations were present in gyrA, gyrB, parC, and/or parE genes among 34 of the 102 naladixic acid-resistant isolates. Two isolates contained class 1 integrons carrying aadA gene conferring streptomycin and spectinomycin resistance. The findings suggest that many antimicrobial agents commonly used in companion animals may not constitute appropriate therapy for canine pseudomonas infections. PMID:18395369

  14. Detection of Metallo-β-Lactamase Producing Pseudomonas aeruginosa Isolated from Public and Private Hospitals in Baghdad, Iraq.

    PubMed

    Al-Charrakh, Alaa H; Al-Awadi, Salwa J; Mohammed, Ahmed S

    2016-02-01

    Metallo-β-lactamase (MBL) producing Pseudomonas aeruginosa has been reported to be an important nosocomial infection. Its intrinsic and acquired resistance to various antimicrobial agents and its ability to develop multidrug resistance imposes a serious therapeutic problem. Different clinical samples were collected from public and private hospitals in Baghdad city, Iraq. Bacterial identification was done using conventional cultural, biochemical tests, and VITEk 2 system. Minimum inhibitory concentration (MIC) testing was performed using VITEK 2 automated system. Each P. aeruginosa isolates showed resistance to Carbapenems (Imipenem and Meropenem) were subjected to Imipenem-EDTA combined disc synergy test (CDST) to investigate the production of MBL (confirmative test). The presence of bla-genes encoded IMP, VIM, and SPM-1 was detected by conventional PCR technique. A total of 75 P. aeruginosa isolates were isolated, 16 (21.3%) were able to grow on MacConkey agar supplemented with Meropenem 4mg/L (MMAC). The MIC of different antibiotics showed that 6 (37.5 %) isolates were Carbapenem resistant, MIC ≥16 µg/ml while 4 (25%) isolates appear to be MBL producer using CDST test. PCR assay revealed that 3 (50%), 1 (16.6%) of the carbapenem resistant isolates harbored blaIMP, blaSPM-1 genes, respectively. blaVIM gene was not detected in this study. The prevalence of multi-drug resistant P. aeruginosa isolates especially Carbapenem resistant bacteria was increased in Baghdad province. The blaIMP was the predominant among the MBLs genes in P. aeruginosa in this study. PMID:26997597

  15. Identification and Characterization of Metallo-β-Lactamases Producing Pseudomonas aeruginosa Clinical Isolates in University Hospital from Zanjan Province, Iran

    PubMed Central

    Doosti, Masoumeh; Ramazani, Ali; Garshasbi, Maryam

    2013-01-01

    Background: Infectious by Pseudomonas aeruginosa has spread worldwide and metallo-beta-lactamases (MBL) are being reported with increasing frequency. The aim of this study was to investigate the antibiotic susceptibility and distribution of blaVIM and blaIMP genes in P. aeruginosa isolates from Zanjan Province of Iran. Methods: A total of 70 P. aeruginosa isolates were identified from patients admitted at intensive care units. The antimicrobial susceptibility was tested by disk diffusion (Kirby-Bauer) method and for production of MBL using double-disk synergy test (DDST). After DNA extraction, the presence of blaVIM and blaIMP genes and class 1 integron were detected by PCR. RESULTS: Most of the isolates were resistant to meropenem, cefotaxime and imipenem (IPM). Also, 44/70 (62.85%) IPM resistant isolates were confirmed by DDST. Of the 44 clinical isolates, 41 (93%) isolates showed MIC≥4 µg/ml for IPM. Based on the DDST results, 36 (87.8%) were confirmed to be MBL producers. PCR amplification showed that 23/41 (56%) carried blaVIM and 10/41 (24.3%) possessed blaIMP gene. Also, 31/44 (70.5%) isolates contained class 1 integron gene. Conclusion: Our results highlight that the genes for Verona integron-encoded metallo-β-lactamase, IPM β-lactamases and class 1 integrons were predominantly present among the IPM-resistant P. aeruginosa tested in our province and also the frequency of blaVIM type is higher than blaIMP. This is the first report of P. aeruginosa strains producing blaIMP with high frequency from Zanjan province of Iran. PMID:23748890

  16. Epidemiology and virulence of VIM-4 metallo-beta-lactamase-producing Pseudomonas aeruginosa isolated from burn patients in eastern Algeria.

    PubMed

    Meradji, Samah; Barguigua, Abouddihaj; Bentakouk, Mohamed Cherif; Nayme, Kaotar; Zerouali, Khalid; Mazouz, Dekhil; Chettibi, Houria; Timinouni, Mohammed

    2016-06-01

    In this study, we investigated the prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in burn patients from eastern Algeria, CRPA virulence factors and the molecular epidemiology of CRPA. The overall prevalence of CRPA was 48.38%. Seven (46.66%) isolates were metallo-β-lactamases (MBL) producers and contained the MBL genes blaVIM-4 (n=6) and blaVIM-2 (n=1). Risk factors for CRPA infection were urinary catheter use and intubation (p=0.008). A high percentage of virulence factors (86.6% of these isolates were able to produce protease; 73.3% of isolates has DNase; and 66.6% were haemolysin positive) was observed in CRPA isolates. Among the seven MBL-producing isolates, four had the same clonal profile. The class 1 integrons, which contained the aadA7 gene cassette, were detected in six isolates. The 16SrRNA methylase gene, rmtB, was detected in one strain. All CRPA isolates were biofilm formers. A study on the kinetics of biofilm production revealed that biofilm production increased when the concentration of imipenem or ciprofloxacin and the incubation time increased. This is the first study to report the presence of VIM-4-producing P. aeruginosa from North Africa and also of the high prevalence of CRPA isolates. Based on our study of burn unit patients, the high percentage of P. aeruginosa with virulence factors and multi-drug resistance is alarming. PMID:27156788

  17. Effect of airway Pseudomonas aeruginosa isolation and infection on steady-state bronchiectasis in Guangzhou, China

    PubMed Central

    Guan, Wei-Jie; Gao, Yong-Hua; Xu, Gang; Lin, Zhi-Ya; Tang, Yan; Li, Hui-Min; Li, Zhi-Min; Zheng, Jin-Ping

    2015-01-01

    Background Current status of Pseudomonas aeruginosa (PA) infection in clinically stable bronchiectasis in mainland China remains unclear. Objective To compare the inflammation and lung function impairment in bronchiectasis patients isolated or infected with PA, potentially pathogenic microorganisms (PPMs) and commensals, and to identify factors associated with PA isolation and infection. Methods Patients with steady-state bronchiectasis and healthy subjects were recruited. Peripheral blood and sputum were sampled to determine inflammatory markers and bacterial loads in steady-state bronchiectasis and health. Spirometry and diffusing capacity were also measured. Results We enrolled 144 bronchiectasis patients and 23 healthy subjects. PA isolation and infection accounted for 44 and 39 patients, who demonstrated significant inflammatory responses and markedly impaired spirometry, but not diffusing capacity, compared with healthy subjects and patients isolated with other PPMs and commensals (all P<0.05). Except for heightened sputum inflammatory responses, there were no notable differences in serum inflammation and lung function as with the increased density of PA. Female gender [odds ratio (OR): 3.10 for PA isolation; OR: 3.74 for PA infection], 4 or more exacerbations within 2 years (OR: 3.74 for PA isolation, OR: 2.95 for PA infection) and cystic bronchiectasis (OR: 3.63 for PA isolation, OR: 4.47 for PA infection) were the factors consistently associated with PA isolation and infection. Conclusions PA elicits intense inflammation and lung function impairment in steady-state bronchiectasis. The density of PA does not correlate with most clinical indices. PA infection is associated with females, frequent exacerbations and cystic bronchiectasis. PMID:25973228

  18. Antimicrobial susceptibility of Pseudomonas aeruginosa isolates from dogs with otitis externa.

    PubMed

    Mekić, S; Matanović, K; Šeol, B

    2011-07-30

    Pseudomonas aeruginosa is a common cause of otitis externa in dogs, and treatment of these infections is becoming problematic because of the increasing number of multiresistant strains. The aim of the present study was to compare the in vitro activities of cefepime, ceftazidime, enrofloxacin, ciprofloxacin, gentamicin and ticarcillin/clavulanic acid against 104 strains of P aeruginosa isolated from dogs with otitis externa. Antimicrobial susceptibility and minimum inhibitory concentrations, in µg/ml, were evaluated by the E test (bioMérieux). The most active compound was ceftazidime, with 100 per cent efficiency. The majority of tested strains were susceptible to ticarcillin/clavulanic acid (89.4 per cent), followed by ciprofloxacin (88.5 per cent) and cefepime (60.6 per cent). The highest resistance was observed to enrofloxacin (51.9 per cent) and gentamicin (43.3 per cent). Large numbers of strains were intermediately susceptible to antibiotics registered for use in veterinary medicine in Croatia--enrofloxacin (47.1 per cent) and gentamicin (41.3 per cent). PMID:21742683

  19. Antibiotic and metal resistance in a ST395 Pseudomonas aeruginosa environmental isolate: A genomics approach.

    PubMed

    Teixeira, Pedro; Tacão, Marta; Alves, Artur; Henriques, Isabel

    2016-09-15

    We analyzed the resistome of Pseudomonas aeruginosa E67, an epiphytic isolate from a metal-contaminated estuary. The aim was to identify genetic determinants of resistance to antibiotics and metals, assessing possible co-selection mechanisms. Identification was based on phylogenetic analysis and average nucleotide identity value calculation. MLST affiliated E67 to ST395, previously described as a high-risk clone. Genome analysis allowed identifying genes probably involved in resistance to antibiotics (e.g. beta-lactams, aminoglycosides and chloramphenicol) and metals (e.g. mercury and copper), consistent with resistance phenotypes. Several genes associated with efflux systems, as well as genetic determinants contributing to gene motility, were identified. Pseudomonas aeruginosa E67 possesses an arsenal of resistance determinants, probably contributing to adaptation to a polluted ecosystem. Association to mobile structures highlights the role of these platforms in multi-drug resistance. Physical links between metal and antibiotic resistance genes were not identified, suggesting a predominance of cross-resistance associated with multidrug efflux pumps. PMID:27371958

  20. 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

  1. Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

    PubMed

    Walther-Rasmussen, J; Johnsen, A H; Høiby, N

    1999-07-01

    AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms. PMID:10406957

  2. The Pseudomonas aeruginosa Lipid A Deacylase: Selection for Expression and Loss within the Cystic Fibrosis Airway

    PubMed Central

    Ernst, Robert K.; Adams, Kristin N.; Moskowitz, Samuel M.; Kraig, Gretchen M.; Kawasaki, Kiyoshi; Stead, Christopher M.; Trent, M. Stephen; Miller, Samuel I.

    2006-01-01

    Lipopolysaccharide (LPS) is the major surface component of gram-negative bacteria, and a component of LPS, lipid A, is recognized by the innate immune system through the Toll-like receptor 4/MD-2 complex. Pseudomonas aeruginosa, an environmental gram-negative bacterium that opportunistically infects the respiratory tracts of patients with cystic fibrosis (CF), can synthesize various structures of lipid A. Lipid A from P. aeruginosa strains isolated from infants with CF has a specific structure that includes the removal of the 3 position 3-OH C10 fatty acid. Here we demonstrate increased expression of the P. aeruginosa lipid A 3-O-deacylase (PagL) in isolates from CF infants compared to that in environmental isolates. PagL activity was increased in environmental isolates by growth in medium limited for magnesium and decreased by growth at low temperature in laboratory-adapted strains of P. aeruginosa. P. aeruginosa PagL was shown to be an outer membrane protein by isopycnic density gradient centrifugation. Heterologous expression of P. aeruginosa pagL in Salmonella enterica serovar Typhimurium and Escherichia coli resulted in removal of the 3-OH C14 fatty acid from lipid A, indicating that P. aeruginosa PagL recognizes either 3-OH C10 or 3-OH C14. Finally, deacylated lipid A species were not observed in some clinical P. aeruginosa isolates from patients with severe pulmonary disease, suggesting that loss of PagL function can occur during long-term adaptation to the CF airway. PMID:16352835

  3. 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

  4. Complete Genome Sequence of Pseudomonas aeruginosa PA1, Isolated from a Patient with a Respiratory Tract Infection

    PubMed Central

    Lu, Shuguang; Le, Shuai; Li, Gang; Shen, Mengyu; Tan, Yinling; Zhao, Xia; Wang, Jing; Shen, Wei; Guo, Keke; Yang, Yuhui; Zhu, Hongbin; Li, Shu; Li, Ming; Zhu, Junmin; Rao, Xiancai

    2015-01-01

    We report the 6,498,072-bp complete genome sequence of Pseudomonas aeruginosa PA1, which was isolated from a patient with a respiratory tract infection in Chongqing, People's Republic of China. Whole-genome sequencing was performed using single-molecule real-time (SMRT) technology, and de novo assembly revealed a single contig with 396-fold sequence coverage. PMID:26659688

  5. Matrix isolation and computational study of isodifluorodibromomethane (F2CBr-Br): A route to Br2 formation in CF2Br2 photolysis

    NASA Astrophysics Data System (ADS)

    George, Lisa; Kalume, Aimable; El-Khoury, Patrick Z.; Tarnovsky, Alexander; Reid, Scott A.

    2010-02-01

    The photolysis products of dibromodifluoromethane (CF2Br2) were characterized by matrix isolation infrared and UV/Visible spectroscopy, supported by ab initio calculations. Photolysis at wavelengths of 240 and 266 nm of CF2Br2:Ar samples (˜1:5000) held at ˜5 K yielded iso-CF2Br2 (F2CBrBr), a weakly bound isomer of CF2Br2, which is characterized here for the first time. The observed infrared and UV/Visible absorptions of iso-CF2Br2 are in excellent agreement with computational predictions at the B3LYP/aug-cc-pVTZ level. Single point energy calculations at the CCSD(T)/aug-cc-pVDZ level on the B3LYP optimized geometries suggest that the isoform is a minimum on the CF2Br2 potential energy surface, lying some 55 kcal/mol above the CF2Br2 ground state. The energies of various stationary points on the CF2Br2 potential energy surface were characterized computationally; taken with our experimental results, these show that iso-CF2Br2 is an intermediate in the Br+CF2Br→CF2+Br2 reaction. The photochemistry of the isoform was also investigated; excitation into the intense 359 nm absorption band resulted in isomerization to CF2Br2. Our results are discussed in view of the rich literature on the gas-phase photochemistry of CF2Br2, particularly with respect to the existence of a roaming atom pathway leading to molecular products.

  6. Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia.

    PubMed

    Cigana, Cristina; Bernardini, Francesca; Facchini, Marcella; Alcalá-Franco, Beatriz; Riva, Camilla; De Fino, Ida; Rossi, Alice; Ranucci, Serena; Misson, Pauline; Chevalier, Eric; Brodmann, Maj; Schmitt, Michel; Wach, Achim; Dale, Glenn E; Obrecht, Daniel; Bragonzi, Alessandra

    2016-08-01

    The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections. PMID:27297477

  7. Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates.

    PubMed

    Karatuna, O; Yagci, A

    2010-12-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe respiratory infections. The pathogenesis of these infections is multifactorial and the production of many virulence factors is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. The two well defined QS systems in P. aeruginosa, the las and rhl systems, rely on N-acyl homoserine lactone signal molecules, also termed autoinducers. We assessed the activity of QS-dependent virulence factors (including elastase, alkaline protease, pyocyanin and biofilm production) in respiratory isolates of P. aeruginosa and their relationship with antimicrobial susceptibility. We identified sixteen isolates displaying impaired phenotypic activity; among them, eleven isolates were also defective in autoinducer production, and therefore considered QS-deficient. Six of the QS-deficient isolates failed to amplify one or more of the four QS regulatory genes (lasI, lasR, rhlI, rhlR) with PCR: one isolate was negative for rhlR, two isolates were negative for rhlI and rhlR and three isolates were negative for all four genes. The isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials and statistically significant correlations were observed between the lack of elastase production and resistance to piperacillin and ceftazidime; between failure in alkaline protease production and resistance to tobramycin, piperacillin, piperacillin-tazobactam, cefepime, imipenem and ciprofloxacin; and between failure in pyocyanin production and resistance to amikacin, tobramycin, ceftazidime, ciprofloxacin and ofloxacin. The results obtained indicate that, despite the pivotal role of QS in the pathogenesis of P. aeruginosa respiratory infections, QS-deficient strains are still capable of causing infections and tend to be less susceptible to antimicrobials. PMID:20132256

  8. Biosurfactant production by Pseudomonas aeruginosa DSVP20 isolated from petroleum hydrocarbon-contaminated soil and its physicochemical characterization.

    PubMed

    Sharma, Deepak; Ansari, Mohammad Javed; Al-Ghamdi, Ahmad; Adgaba, Nuru; Khan, Khalid Ali; Pruthi, Vikas; Al-Waili, Noori

    2015-11-01

    Among 348 microbial strains isolated from petroleum hydrocarbon-contaminated soil, five were selected for their ability to produce biosurfactant based on battery of screening assay including hemolytic activity, surface tension reduction, drop collapse assay, emulsification activity, and cell surface hydrophobicity studies. Of these, bacterial isolate DSVP20 was identified as Pseudomonas aeruginosa (NCBI GenBank accession no. GQ865644) based on biochemical characterization and the 16S rDNA analysis, and it was found to be a potential candidate for biosurfactant production. Maximum biosurfactant production recorded by P. aeruginosa DSVP20 was 6.7 g/l after 72 h at 150 rpm and at a temperature of 30 °C. Chromatographic analysis and high-performance liquid chromatography-mass spectrometry (HPLC-MS) revealed that it was a glycolipid in nature which was further confirmed by nuclear magnetic resonance (NMR) spectroscopy. Bioremediation studies using purified biosurfactant showed that P. aeruginosa DSVP20 has the ability to degrade eicosane (97%), pristane (75%), and fluoranthene (47%) when studied at different time intervals for a total of 7 days. The results of this study showed that the P. aeruginosa DSVP20 and/or biosurfactant produced by this isolate have the potential role in bioremediation of petroleum hydrocarbon-contaminated soil. PMID:26146372

  9. An antimicrobial diketopiperazine alkaloid and co-metabolites from an endophytic strain of Gliocladium isolated from Strychnos cf. toxifera.

    PubMed

    Koolen, Hector Henrique Ferreira; Soares, Elzalina Ribeiro; Silva, Felipe Moura Araújo da; Souza, Antonia Queiroz Lima de; Medeiros, Lívia Soman de; Filho, Edson Rodrigues; Almeida, Richardson Alves de; Ribeiro, Ismael Alexandre; Pessoa, Cláudia do Ó; Morais, Manoel Odorico de; Costa, Patrícia Marçal da; Souza, Afonso Duarte Leão de

    2012-11-01

    From an endophytic strain of Gliocladium sp. isolated from the Amazonian plant Strychnos cf. toxifera, we obtained the diketopiperazine alkaloid cyclo-(glycyl-L-tyrosyl)-4,4-dimethylallyl ether (1), the steroids ergosterol (2), ergosterol peroxide (3), cerevisterol (4) and the citric acid (5). The AcOEt extract of the fermented broth by Gliocladium sp. showed potent activity against the cancer cell lines MDA-MB435 (human breast cancer cells), HCT-8 (human colorectal cancer cells) and SF-295 (human glioblastoma cancer cells). Compound 1 exhibited a strong antimicrobial activity against Micrococcus luteus at a concentration of 43.4 µM. PMID:22117164

  10. Tanjungides A and B: New Antitumoral Bromoindole Derived Compounds from Diazona cf formosa. Isolation and Total Synthesis

    PubMed Central

    Murcia, Carmen; Coello, Laura; Fernández, Rogelio; Martín, María Jesús; Reyes, Fernando; Francesch, Andrés; Munt, Simon; Cuevas, Carmen

    2014-01-01

    Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey’s analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines. PMID:24566261

  11. 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

  12. Role of Adherence in the Pathogenesis of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis Patients

    PubMed Central

    Woods, Donald E.; Bass, Joe A.; Johanson, W. G.; Straus, David C.

    1980-01-01

    A correlation has been demonstrated between the in vitro adherence of Pseudomonas aeruginosa to upper respiratory tract epithelium and colonization of the respiratory tract by this organism. Twenty patients with cystic fibrosis (CF) and 20 age-matched controls were examined in this study. All of the CF patients but none of the controls were colonized with P. aeruginosa at the time of study. P. aeruginosa adherence to isolated epithelial cells, as determined by an in vitro assay, was 19.1 ± 1.1 bacteria per buccal epithelial cell in the CF patients and 2.3 ± 0.3 bacteria per cell in the controls (P < 0.01). P. aeruginosa strains of the mucoid colony type adhered in significantly lower numbers to buccal epithelial cells than did strains of the rough colony type (1.8 + 0.1 versus 24.8 ± 0.9, P < 0.001). This difference might explain the common observation that the initial pseudomonas colonization of the respiratory tract of CF patients is due to organisms of the rough colony type. We have further demonstrated that increased P. aeruginosa adherence in vitro varies directly with the loss of a protease-sensitive glycoprotein, fibronectin, from the cell surface, as well as increased levels of salivary proteases in CF patients. When examined by a direct radioimmune binding assay, buccal cells from CF patients possessed only 17% of the total cell surface fibronectin present on similar cells obtained from controls. Salivary protease levels, as measured by 125I release from an 125I-labeled insoluble fibrin matrix, were increased about threefold in CF patients versus controls. Thus, colonization of the respiratory tract by P. aeruginosa in CF patients correlates well with buccal cell adherence of this organism; increased adherence is associated with decreased amounts of fibronectin on respiratory epithelial cell surfaces and increased levels of salivary proteases. PMID:7014444

  13. Characterization of Pseudomonas aeruginosa isolates from dogs and cats in Japan: current status of antimicrobial resistance and prevailing resistance mechanisms.

    PubMed

    Harada, Kazuki; Arima, Sayuri; Niina, Ayaka; Kataoka, Yasushi; Takahashi, Toshio

    2012-02-01

    Seventy-three Pseudomonas aeruginosa isolates were collected from dogs and cats in Japan to investigate antimicrobial susceptibility and resistance mechanisms to anti-pseudomonal agents. Resistance rates against orbifloxacin, enrofloxacin, ciprofloxacin, cefotaxime, aztreonam and gentamicin were 34.2, 31.5, 20.5, 17.8, 12.3 and 4.1%, respectively. The degree of resistance to cefotaxime, orbifloxacin, and enrofloxacin was greatly affected by efflux pump inhibitors, indicating overexpression of efflux pump contributes to these resistances. Notably, orbifloxacin and enrofloxacin resistance was observed even in isolates without mutations in the target sites. This is the first report on cephalosporin- and fluoroquinolone-resistant isolates of P. aeruginosa from Japanese companion animals. PMID:22188523

  14. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam

    PubMed Central

    2013-01-01

    Background 16S rRNA methylase-producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed clinical strains of 16S rRNA methylase-producing Acinetobactor baumannii and Pseudomonas aeruginosa obtained from clinical isolates in medical settings in Vietnam. Methods From 2008 to 2011, 101 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam. Antimicrobial susceptibilities were determined using the microdilution method and epidemiological analysis was performed by pulsed-field gel electrophoresis and MLST. Genes encoding the 16S rRNA methylases, OXAs and CTX-Ms were analyzed by PCR and sequence analysis. Results 16S rRNA methylase-producing Gram-negative pathogens were detected in two hospitals in Vietnam. Of the 101 clinical isolates of A. baumannii and the 15 of P. aeruginosa isolated from two ICUs in these hospitals, 72 (71.3%) were highly resistant to amikacin, arbekacin and gentamicin, with MICs greater than 1,024 mg/L. The 16S rRNA methylases ArmA and RmtB were produced by 61 and 9 isolates of A. baumannii, respectively, and RmtB was produced by 2 isolates of P. aeruginosa. Moreover, 52 of the A. baumannii isolates producing 16S rRNA methylases harbored both blaOXA-23-like and blaOXA-51-like genes. Most A. baumannii isolates producing 16S rRNA methylase obtained in hospital A in Hanoi were ST91 and ST231, whereas most from hospital B in Ho Chi Minh City were ST136, ST195, and ST254. The two P. aeruginosa isolates harboring rmtB showed different patterns on PFGE, one each corresponding to ST217 and ST313. Conclusions Gram-negative bacteria producing the 16S rRNA methylases ArmA and RmtB are emerging in medical settings in Vietnam. A. baumannii isolates in northern and southern regions of Vietnam may be of different lineages. PMID:23721359

  15. Isolation and identification of a novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110

    PubMed Central

    Mobarak-Qamsari, E; Kasra-Kermanshahi, R; Moosavi-nejad, Z

    2011-01-01

    Background and Objectives Lipases are particularly important due to the fact that they specifically hydrolyze acyl glycerol, oils and greases, which is of great interest for different industrial applications. Materialst and Methods In this study, several lipase-producing bacteria were isolated from wastewater of an oil processing plant. The strain possessing the highest lipase activity was identified both biochemically and sequencing of 16S rRNA gene. Then we increase lipase activity by improving conditions of production medium. Also, lipase from this strain was preliminarily characterized for use in industrial application. Results The 16S rRNA sequensing revealed it as a new strain of Pseudomonas aeruginosa and the type strain was KM110. An overall 3-fold enhanced lipase production (0.76 U mL−1) was achieved after improving conditions of production medium. The olive oil and peptone was found to be the most suitable substrate for maximum enzyme production. Also the enzyme exhibited maximum lipolytic activity at 45°C where it was also stably maintained. At pH 8.0, the lipase had the highest stability but no activity. It was active over a pH range of 7.0–10.0. The lipase activity was inhibited by Zn2+ & Cu2+ (32 and 27%, respectively) at 1mM. The enzyme lost 29% of its initial activity in 1.0% SDS concentration, whereas, Triton X-100, Tween-80 & DMSO did not significantly inhibit lipase activity. Conclusions Based on the findings of present study, lipase of P. aeruginosa KM110 is a potential alkaline lipase and a candidate for industrial applications such as detergent, leather and fine chemical industries. PMID:22347589

  16. [The annual changes in antimicrobial susceptibility test results of Pseudomonas aeruginosa isolates from the Kinki district].

    PubMed

    Fukuda, Saori; Komatsu, Masaru; Nakamura, Tatuya; Jikimoto, Takumi; Nishio, Hisaaki; Yamasaki, Katsutoshi; Satoh, Kaori; Toda, Hirofumi; Orita, Tamaki; Sueyoshi, Noriyuki; Kita, Machiko; Nishi, Isao; Akagi, Masahiro; Higuchi, Takeshi; Kofuku, Tomomi; Nakai, Isako; Ono, Tamotsu; Kida, Kaneyuki; Ohama, Masanobu; Watari, Hideo; Shimura, Satoshi; Niki, Makoto; Kuchibiro, Tomokazu; Wada, Yasunao

    2016-04-01

    A study was conducted of the 1,225 Pseudomonas aeruginosa strains that were isolated at 20 medical institutions in the Kinki district between 2011 and 2013 to determine their antimicrobial susceptibility and to characterize the strains of multidrug-resistant Pseudomonas aeruginosa (MDRP) and the metallo-β-lactamase (MBL) -producing strains. The MIC50/MIC90 values (μg/mL) of the various antimicrobial agents were as follows: imipenem, 2/>8; meropenem, 1/>8; doripenem, 0.5/8; biapenem, 1/>8; tazobactam/piperacillin, 8/>64; piperacillin, 8/>64; sulbactam/cefoperazone, 8/64; cefepime, 4/16; cefozopran, 2/>16; aztreonam, 8/>16; amikacin, 4/16; levofloxacin, 1/>4; and ciprofloxacin, 0.25/>2. From the viewpoint of the annual changes in the susceptibility rates (according to the CLSI guidelines [M100-S22]), the susceptibility to tazobactam/piperacillin, piperacillin, cefepime, cefozopran and aztreonam decreased in 2013. On the other hand, two antimicrobial agents showed high susceptibility rates each year; amikacin (94.0-95.6%) showed the highest rate, followed by doripenem (80.3-82.6%). With the exception of amikacin, there were substantial inter-institutional differences in antimicrobial susceptibility. In comparison to the previous CLSI guidelines (M100-S21), the new CLSI guidelines (M100-S22) on the use of carbapenems and penicillins show that the MIC80 has been affected. The MDRP detection rates in 2011, 2012 and 2013 were 1.8% (8 strains), 1.8% (8 strains), and 2.8% (10 strains), respectively. The MBL detection rates were as follows: bla(VIM-2), 0.2% (1 strain) in 2011; bla(IMP-1), 0.9% (4 strains) in 2012, and 1.7% (6 strains, including bla(IMP-1) [3 strains], bla(IMP-2) [2 strains] and bla(VIM-2) [1 strain]) in 2013. PMID:27544978

  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. 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

  19. A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence

    PubMed Central

    Sall, Khady Mayebine; Casabona, Maria Guillermina; Bordi, Christophe; Huber, Philippe; de Bentzmann, Sophie; Attrée, Ina; Elsen, Sylvie

    2014-01-01

    Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 3′ end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1-T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor. PMID:24780952

  20. PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

    PubMed Central

    Brannon, Mark K.; Dasgupta, Nandini; Pier, Miyuki; Sgambati, Nicole; Miller, Amanda K.; Selgrade, Sara E.; Miller, Samuel I.; Denton, Miles; Conway, Steven P.; Johansen, Helle K.; Høiby, Niels

    2012-01-01

    Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosa isolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa. PMID:22106224

  1. Epidemiology of Pseudomonas aeruginosa in a tertiary referral teaching hospital.

    PubMed

    Bradbury, R S; Champion, A C; Reid, D W

    2009-10-01

    A genotypically indistinguishable strain of Pseudomonas aeruginosa (Australian epidemic strain III: AES III) has previously been found in a proportion of adults with cystic fibrosis (CF) in Tasmania, Australia. The aim of this study was to identify a source of these infections within the major tertiary referral hospital for the State of Tasmania, and to determine if this strain could be isolated from settings other than the CF lung. A total of 120 isolates of P. aeruginosa were collected from clinical and environmental sources within the hospital and from environmental locations in the hospital vicinity. These isolates were genotyped by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. Confirmation of similar genotypes identified by RAPD-PCR was performed using pulsed-field gel electrophoresis with restriction enzyme SpeI. AES III was not recovered from any source other than the respiratory secretions of CF patients. P. aeruginosa in the non-CF settings was found to be panmictic, and no cross-infection or acquisition of hospital environment strains by patients was observed. PMID:19699556

  2. 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

  3. 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

  4. Antibiotic resistance pattern of Pseudomonas aeruginosa isolated from urine samples of Urinary Tract Infections patients in Karachi, Pakistan

    PubMed Central

    Shah, Dania Aijaz; Wasim, Shehnaz; Essa Abdullah, Farhan

    2015-01-01

    Objective: The aim of this study was to evaluate the antibiotic resistance pattern of Psedomonas aeruginosa and its prevalence in patients with urinary tract infections (UTI) for effective treatment in a developing country like Pakistan. Methods: This is an observational study conducted for a period of ten months which ended on December 2013 at the Dr. Essa Laboratory and Diagnostic Centre in Karachi. A total of 4668 urine samples of UTI patients were collected and standard microbiological techniques were performed to identify the organisms in urine cultures. Antibiotic susceptibility testing was performed by Kirby-Bauer technique for twenty five commonly used antimicrobials and then analyzed on SPSS version 17. Results: P. aeruginosa was isolated in 254 cultures (5.4%). The most resistant drugs included Ceclor(100%) and Cefizox (100%) followed by Amoxil/Ampicillin (99.6%), Ceflixime (99.6%), Doxycycline (99.6%), Cefuroxime (99.2%), Cephradine (99.2%), Cotrimoxazole (99.2%), Nalidixic acid (98.8%), Pipemidic acid (98.6%) and Augmentin (97.6%). Conclusion: Emerging resistant strains of Pseudomonas aeruginosa are potentially linked to injudicious use of drugs leading to ineffective empirical therapy and in turn, appearance of even more resistant strains of the bacterium. Therefore, we recommend culture and sensitivity testing to determine the presence of P.aeruginosa prior to specific antimicrobial therapy. PMID:26101487

  5. Potential of Ocimum basilicum L. and Salvia officinalis L. essential oils against biofilms of P. aeruginosa clinical isolates.

    PubMed

    Stojanović-Radić, Z; Pejcić, M; Stojanović, N; Sharifi-Rad, J; Stanković, N

    2016-01-01

    Biofilms are complex communities of microorganisms, responsible for more than 60% of the chronic human infections and they represent one of the leading concerns in medicine. Pseudomonas aeruginosa is human pathogenic bacteria which causes numerous diseases and is known for its ability to produce biofilm. Ocimum basilicum L. (basil) and Salvia officinalis L. (sage) are widely used plants in traditional medicine for the treatment of different conditions. Therefore, the aim of this study was to investigate the potential of basil and sage essential oils against P. aeruginosa biofilm producing strains. The efficacy of two essential oils on P. aeruginosa biofilm forming ability was determined using crystal violet method. Out of 15 strains isolated from different clinical biological samples, two were strong, 11 moderate and one weak biofilm producer. Good efficacy of sage essential oil towards strong and weak biofilm producers, but not of basil essential oil, was observed. In the case of moderate biofilm producers, 81.8% showed lower biofilm production after incubation with the sage oil, while 63.6% showed the reduction of biofilm production after basil essential oil treatment. The obtained results showed high potential of both oils for the treatment of persistent infections caused by Pseudomonas aeruginosa biofilms. PMID:27585258

  6. 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.

  7. Laser irradiation effect on Staphylococcus aureus and Pseudomonas aeruginosa biofilms isolated from venous leg ulcer.

    PubMed

    Baffoni, Marina; Bessa, Lucinda J; Grande, Rossella; Di Giulio, Mara; Mongelli, Matteo; Ciarelli, Antonio; Cellini, Luigina

    2012-10-01

    Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, represent a significant cause of morbidity in developed countries, predominantly in older patients. The aetiology of these wounds is probably multifactorial, but the role of bacteria in their pathogenesis is still unclear. Moreover, the presence of bacterial biofilms has been considered an important factor responsible for wounds chronicity. We aimed to investigate the laser action as a possible biofilm eradicating strategy, in order to attempt an additional treatment to antibiotic therapy to improve wound healing. In this work, the effect of near-infrared (NIR) laser was evaluated on mono and polymicrobial biofilms produced by two pathogenic bacterial strains, Staphylococcus aureus PECHA10 and Pseudomonas aeruginosa PECHA9, both isolated from a chronic venous leg ulcer. Laser effect was assessed by biomass measurement, colony forming unit count and cell viability assay. It was shown that the laser treatment has not affected the biofilms biomass neither the cell viability, although a small disruptive action was observed in the structure of all biofilms tested. A reduction on cell growth was observed in S. aureus and in polymicrobial biofilms. This work represents an initial in vitro approach to study the influence of NIR laser treatment on bacterial biofilms in order to explain its potentially advantageous effects in the healing process of chronic infected wounds. PMID:22182280

  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. Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs

    PubMed Central

    Malone, Jacob G

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis (CF) lung infections. Over many years of chronic lung colonization, P. aeruginosa undergoes extensive adaptation to the lung environment, evolving both toward a persistent, low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs. These lung-adapted P. aeruginosa strains include the small colony variants (SCVs), small, autoaggregative isolates that show enhanced biofilm formation, strong attachment to surfaces, and increased production of exopolysaccharides. Their appearance in the sputum of CF patients correlates with increased resistance to antibiotics, poor lung function, and prolonged persistence of infection, increasing their relevance as a subject for clinical investigation. The evolution of SCVs in the CF lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic-di-GMP, with increased cyclic-di-GMP levels shown to be responsible for the SCV phenotype in a number of different CF lung isolates. Here, we review the current state of research in clinical P. aeruginosa SCVs. We will discuss the phenotypic characteristics underpinning the SCV morphotype, the clinical implications of lung colonization with SCVs, and the molecular basis and clinical evolution of the SCV phenotype in the CF lung environment. PMID:26251621

  10. Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.

    PubMed

    Vidya, P; Smith, L; Beaudoin, T; Yau, Y C; Clark, S; Coburn, B; Guttman, D S; Hwang, D M; Waters, V

    2016-01-01

    Early eradication treatment with inhaled tobramycin is successful in the majority of children with cystic fibrosis (CF) with incident Pseudomonas aeruginosa infection. However, in 10-40 % of cases, eradication fails and the reasons for this are poorly understood. The purpose of this study was to determine whether specific microbial characteristics could explain eradication treatment failure. This was a cross-sectional study of CF patients (aged 0-18 years) with incident P. aeruginosa infection from 2011 to 2014 at the Hospital for Sick Children, Toronto, Canada. Phenotypic assays were done on all incident P. aeruginosa isolates, and eradicated and persistent isolates were compared using the Mann-Whitney test or the two-sided Chi-square test. A total of 46 children with CF had 51 incident P. aeruginosa infections. In 72 % (33/46) of the patients, eradication treatment was successful, while 28 % failed eradication therapy. Persistent isolates were less likely to be motile, with significantly less twitch motility (p=0.001), were more likely to be mucoid (p=0.002), and more likely to have a tobramycin minimum inhibitory concentration (MIC) ≥ 128 μg/mL (p=0.02) compared to eradicated isolates. Although biofilm production was similar, there was a trend towards more persistent isolates with deletions in quorum-sensing genes compared with eradicated isolates (p=0.06). Initial acquisition of P. aeruginosa with characteristics of chronic infection is associated with failure of eradication treatment. PMID:26492874

  11. First Survey of Metallo-β–Lactamase Producers in Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province

    PubMed Central

    Kalantar, Enayatollah; Torabi, Vahideh; Salimizand, Heiman; Soheili, Fariborz; Beiranvand, Soheila; Soltan Dallal, Mohammad Mehdi

    2012-01-01

    Background Treatment of infectious diseases is becoming more challenging with each passing year. This is especially true for infections caused by Pseudomonas aeruginosa, an opportunistic pathogen with the ability to rapidly develop resistance to multiple classes of antibiotics. Objectives This study was conducted to determine the prevalence of metallo-β-lactamase (MBL)–producing strains among multidrug-resistant P. aeruginosa strains isolated from burn patients. Materials and Methods The isolates were identified, tested for susceptibility to various antimicrobial agents, and screened for the presence of MβLs by using the double-disk synergy test. The minimal inhibitory concentration of imipenem was determined by microplate broth dilution method on Mueller-Hinton agar. To detect VIM, SIM, and GIM MBLs, the isolates were subjected to polymerase chain reaction. Results In this study, we identified 100 P. aeruginosa isolates from 176 clinical specimens obtained from burn patients. The isolates showed maximum resistance to ampicillin (100%), ceftazidime (94%), and ceftriaxone (89%). The CLSI-MBL phenotypic test showed that of the 100 P. aeruginosa isolates, 22 (22%) were positive for MBL production in the double-disk synergy test. Of the 22 MBL-positive P. aeruginosa isolates, 8 were resistant to imipenem. PCR analysis showed that 8 isolates were positive for blaVIM1. The other genes blaSIM1 and blaGIM1 were not detected. Conclusions The study results demonstrate the serious therapeutic threat of the spread of MBL producers among P. aeruginosa populations. Metallo-β-lactamases were detected in 22% of imipenem-resistant P. aeruginosa isolates. Early detection and infection-control practices are the best antimicrobial strategies for this organism; therefore, systematic surveillance to detect MBL producers is necessary. PMID:24624147

  12. Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

    PubMed Central

    2013-01-01

    Background The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. Methods We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. Results Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. Conclusion Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies. PMID:24088260

  13. Presence of exoY, exoS, exoU and exoT genes, antibiotic resistance and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran

    PubMed Central

    Azimi, Somayeh; Kafil, Hossein Samadi; Baghi, Hossein Bannazadeh; Shokrian, Saeed; Najaf, Khadijeh; Asgharzadeh, Mohammad; Yousefi, Mehdi; Shahrivar, Firooz; Aghazadeh, Mohammad

    2016-01-01

    Background: Pseudomonas aeruginosa, as Gram-negative rod bacilli, has an important role in human infection. In the present study we aimed to investigate the presence of exo genes and biofilm production among Pseudomonas aeruginosa isolates in Northwest Iran. Material and methods: 160 isolates of P. aeruginosa were collected and identified by biochemical tests and were characterized for antibiotic resistance. Biofilm production was evaluated by microtiter plate assay and the presence of exo genes was evaluated by allele-specific PCR (polymerase chain reaction). Chi-square test was used for statistical analysis. Results: The most effective antibiotics against isolates were colistin and polymyxin B. 87% of the isolates were biofilm producers of which 69% were strongly biofilm producers. 55% of the isolates carried exoY, 52% of the isolates carried exoU, and 26.3% and 5% carried exoS and exoT, respectively. Conclusion: Our findings showed different distribution of exo genes in clinical isolates of P. aeruginosa in Northwest Iran. ExoS and exoU were more prevalent in non-biofilm producers and exoY was more prevalent in biofilm producer isolates. These results might indicate the importance of exoY in biofilm production of Pseudomonas aeruginosa. PMID:26958458

  14. Additional insights on the bastadins: isolation of analogues from the sponge Ianthella cf. reticulata and exploration of the oxime configurations.

    PubMed

    Calcul, Laurent; Inman, Wayne D; Morris, Alexi A; Tenney, Karen; Ratnam, Joseline; McKerrow, James H; Valeriote, Frederick A; Crews, Phillip

    2010-03-26

    The focus of this study is on the bastadin class of bromotyrosine derivatives, commonly isolated from Ianthella marine sponges, and is the first report on the secondary metabolites from Ianthella cf. reticulata. Two new bastadins were isolated, (E,Z)-bastadin 19 (1a), a diastereoisomer of the known (E,E)-bastadin 19 (1b), and dioxepine bastadin 3 (2), an unusual dibenzo-1,3-dioxepine. A bastadin NMR database was created and assisted in the structure determination of 1b and 2 and the rapid dereplication of 10 other known compounds including bastadins 2-9 (3-10), 13 (11), and 19 (1a). The geometry of the 2-(hydroxyimino)-N-alkylamide chains, a chemical feature present in all bastadins, was further probed, and new insights regarding the natural oxime configuration are discussed. Bastadins possessing (E,Z)-, (Z,E)-, or (E,E)-dioxime configurations could be artifacts of isolation or storage in solution. Therefore, this point was explored by photochemical and thermal isomerization studies, as well as molecular mechanics calculations. Bastadins 13 (11) and 19 (1a) exhibited moderate inhibition against Trypanosoma brucei, and bastadin 4 (5) was cytotoxic to HCT-116 colon cancer cells. PMID:20102170

  15. Sequence Types 235, 111, and 132 Predominate among Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates in Croatia

    PubMed Central

    Izdebski, Radosław; Butic, Iva; Jelic, Marko; Abram, Maja; Koscak, Iva; Baraniak, Anna; Hryniewicz, Waleria; Gniadkowski, Marek; Tambic Andrasevic, Arjana

    2014-01-01

    A population analysis of 103 multidrug-resistant Pseudomonas aeruginosa isolates from Croatian hospitals was performed. Twelve sequence types (STs) were identified, with a predominance of international clones ST235 (serotype O11 [41%]), ST111 (serotype O12 [15%]), and ST132 (serotype O6 [11%]). Overexpression of the natural AmpC cephalosporinase was common (42%), but only a few ST235 or ST111 isolates produced VIM-1 or VIM-2 metallo-β-lactamases or PER-1 or GES-7 extended-spectrum β-lactamases. PMID:25070098

  16. Resistance to the quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil in Pseudomonas aeruginosa clinical isolates.

    PubMed

    García-Contreras, Rodolfo; Martínez-Vázquez, Mariano; Velázquez Guadarrama, Norma; Villegas Pañeda, Alejandra Guadalupe; Hashimoto, Takahiro; Maeda, Toshinari; Quezada, Héctor; Wood, Thomas K

    2013-06-01

    The quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil inhibit the pathogenicity of the Pseudomonas aeruginosa laboratory strains PA01 and PA14; however, there is no report studying the effectiveness of these compounds for clinical isolates. Therefore, the effect of both quorum quenchers on the production of pyocyanin, elastase and alkaline protease of eight clinical strains from children was evaluated. Although both compounds were in general effective for the attenuation of these factors, three strains resistant to C-30 were found. For 5-fluorouracil, PA01 and some clinical isolates showed resistance for at least one phenotype. PMID:23620228

  17. Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates

    PubMed Central

    Charretier, Yannick; Köhler, Thilo; Cecchini, Tiphaine; Bardet, Chloé; Cherkaoui, Abdessalam; Llanes, Catherine; Bogaerts, Pierre; Chatellier, Sonia; Charrier, Jean-Philippe; Schrenzel, Jacques

    2015-01-01

    Both acquired and intrinsic mechanisms play a crucial role in Pseudomonas aeruginosa antibiotic resistance. Many clinically relevant resistance mechanisms result from changes in gene expression, namely multidrug efflux pump overproduction, AmpC β-lactamase induction or derepression, and inactivation or repression of the carbapenem-specific porin OprD. Changes in gene expression are usually assessed using reverse-transcription quantitative real-time PCR (RT-qPCR) assays. Here, we evaluated label-free Selected Reaction Monitoring (SRM)-based mass spectrometry to directly quantify proteins involved in antibiotic resistance. We evaluated the label-free SRM using a defined set of P. aeruginosa isolates with known resistance mechanisms and compared it with RT-qPCR. Referring to efflux systems, we found a more robust relative quantification of antibiotic resistance mechanisms by SRM than RT-qPCR. The SRM-based approach was applied to a set of clinical P. aeruginosa isolates to detect antibiotic resistance proteins. This multiplexed SRM-based approach is a rapid and reliable method for the simultaneous detection and quantification of resistance mechanisms and we demonstrate its relevance for antibiotic resistance prediction. PMID:25713571

  18. Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates.

    PubMed

    Charretier, Yannick; Köhler, Thilo; Cecchini, Tiphaine; Bardet, Chloé; Cherkaoui, Abdessalam; Llanes, Catherine; Bogaerts, Pierre; Chatellier, Sonia; Charrier, Jean-Philippe; Schrenzel, Jacques

    2015-01-01

    Both acquired and intrinsic mechanisms play a crucial role in Pseudomonas aeruginosa antibiotic resistance. Many clinically relevant resistance mechanisms result from changes in gene expression, namely multidrug efflux pump overproduction, AmpC β-lactamase induction or derepression, and inactivation or repression of the carbapenem-specific porin OprD. Changes in gene expression are usually assessed using reverse-transcription quantitative real-time PCR (RT-qPCR) assays. Here, we evaluated label-free Selected Reaction Monitoring (SRM)-based mass spectrometry to directly quantify proteins involved in antibiotic resistance. We evaluated the label-free SRM using a defined set of P. aeruginosa isolates with known resistance mechanisms and compared it with RT-qPCR. Referring to efflux systems, we found a more robust relative quantification of antibiotic resistance mechanisms by SRM than RT-qPCR. The SRM-based approach was applied to a set of clinical P. aeruginosa isolates to detect antibiotic resistance proteins. This multiplexed SRM-based approach is a rapid and reliable method for the simultaneous detection and quantification of resistance mechanisms and we demonstrate its relevance for antibiotic resistance prediction. PMID:25713571

  19. Characterization of exo-s, exo-u, and alg virulence factors and antimicrobial resistance in Pseudomonas aeruginosa isolated from migratory Egyptian vultures from India.

    PubMed

    Sharma, Pradeep; Faridi, Farah; Mir, Irfan A; Sharma, Sandeep K

    2014-01-01

    This study of Pseudomonas aeruginosa in fecal droppings of migratory Egyptian vultures (Neophron p. percnopterus) revealed eight positive samples (n=25) by a 16S rRNA gene-based PCR in two consecutive winter seasons. Disk diffusion sensitivity testing revealed three multiple antimicrobial resistant (MAR) isolates. Genotypic characterization showed mutually exclusive exo-s and exo-u virulence genes in five and three isolates, respectively, while the alg gene was present in all of the isolates. MAR isolates with virulence genes were detected in both seasons. The Egyptian vultures could potentially be vectors of pathogenic and MAR P. aeruginosa, thereby affecting regional control and preventive measures. PMID:25317261

  20. Plasmid Profile Analysis and bla VIM Gene Detection of Metalo β-lactamase (MBL) Producing Pseudomonas aeruginosa Isolates from Clinical Samples

    PubMed Central

    M, Jeya

    2014-01-01

    Introduction:Pseudomonas aeruginosa is a frequent colonizer of hospitalized patients. They are responsible for serious infections such as meningitis, urological infections, septicemia and pneumonia. Carbapenem resistance of Pseudomonas aeruginosa is currently increasingly reported which is often mediated by production of metallo-β-lactamase (MBL). Multidrug resistant Pseudomonas aeruginosa isolates may involve reduced cell wall permeability, production of chromosomal and plasmid mediated β lactamases, aminoglycosides modifying enzymes and an active multidrug efflux mechanism. Objective: This study is aimed to detect the presence and the nature of plasmids among metallo-β-lactamase producing Pseudomonas aeruginosa isolates. Also to detect the presence of bla VIM gene from these isolates. Materials and Methods: Clinical isolates of Pseudomonas aeruginosa showing the metalo-β-lactamase enzyme (MBL) production were isolated. The MBL production was confirmed by three different methods. From the MBL producing isolates plasmid extraction was done by alkaline lysis method. Plasmid positive isolates were subjected for blaVIM gene detection by PCR method. Results: Two thousand seventy six clinical samples yielded 316 (15.22%) Pseudomonas aeruginosa isolates, out of which 141 (44.62%) were multidrug resistant. Among them 25 (17.73%) were metallo-β-lactamase enzyme producers. Plasmids were extracted from 18 out of 25 isolates tested. Five out of 18 isolates were positive for the blaVIM gene detection by the PCR amplification. Conclusion: The MBL producers were susceptible to polymyxin /colistin with MIC ranging from 0.5 – 2μg/ml. Molecular detection of specific genes bla VIM were positive among the carbapenem resistant isolates. PMID:25120980

  1. 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

  2. Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin.

    PubMed

    Marvig, Rasmus Lykke; Damkiær, Søren; Khademi, S M Hossein; Markussen, Trine M; Molin, Søren; Jelsbak, Lars

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. IMPORTANCE Most bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While

  3. Toxicity of microcystin-LR, isolated from Microcystis aeruginosa, against various insect species.

    PubMed

    Delaney, J M; Wilkins, R M

    1995-06-01

    Microcystin-LR (MC-LR), isolated from the cyanobacterium Microcystis aeruginosa Kuetzing emend. Elenkin strain CCAP 1450/4 was tested for biological activity against four species of insect and the invertebrate Artemia salina. The efficacy of pesticidal activity was compared with various insecticides. The 24 hr LD50 value for third instar diamond-backed moth, Plutella xylostella, on ingestion from a treated leaf surface was 1.0 micrograms cm2, compared with a 72 hr LD50 value for rotenone of 2.0 micrograms cm-2. The 24 hr LD50 values of MC-LR and malathion on intrathoracic injection into adult house flies (Musca domestica) were 0.5 and 3.7 mg kg-1, respectively. MC-LR had no effect on M. domestica when applied topically at dosages up to 32 mg kg-1. MC-LR and malathion gave 24 hr LD50 values of 4.7 and 13.1 mg kg-1, respectively when injected into third instar cotton leafworm (Spodoptera littoralis). In fourth instar cabbage white butterfly larvae (Pieris brassicae) MC-LR injected gave 24 and 48 hr LD50 values of 3.9 and 1.9 mg kg-1, respectively, whilst the 24 and 48 hr LD50 values for carbofuran were 0.4 and 0.3 mg kg-1, respectively. An immersion bioassay with 1-day-old brine shrimp larvae (Artemia salina) gave 24 hr LD50 values of 3.8 micrograms ml-1 for MC-LR and 1.8 micrograms ml-1 for carbofuran. MC-LR has appreciable insect toxicity, comparable to the three insecticides tested. The toxin look 24-48 hr to exert its full lethal effect in insects, much longer than the 1-3 hr it takes in mammals. The potential use of MC-LR as an insecticide is discussed. PMID:7676468

  4. Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry.

    PubMed

    Fleurbaaij, Frank; Kraakman, Margriet E M; Claas, Eric C J; Knetsch, Cornelis W; van Leeuwen, Hans C; van der Burgt, Yuri E M; Veldkamp, Karin Ellen; Vos, Margreet C; Goessens, Wil; Mertens, Bart J; Kuijper, Ed J; Hensbergen, Paul J; Nicolardi, Simone

    2016-06-01

    The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database. It is fast and reliable, and costs are low compared to previous diagnostic approaches. However, the low resolution and dynamic range of the MALDI-TOF profiles have shown limited applicability for the discrimination of different bacterial strains, as achieved with typing based on genetic markers. This is pivotal in cases where certain strains are associated with, e.g., virulence or antibiotic resistance. Ultrahigh resolution MALDI-FTICR MS allows the measurement of small proteins at isotopic resolution and can be used to analyze complex mixtures with increased dynamic range and higher precision than MALDI-TOF MS, while still generating results in a similar time frame. Here, we propose to use ultrahigh resolution 15T MALDI-Fourier transform ion cyclotron resonance (FTICR) MS to discriminate clinically relevant bacterial strains after species identification performed by MALDI-TOF MS. We used a collection of well characterized Pseudomonas aeruginosa strains, featuring distinct antibiotic resistance profiles, and isolates obtained during hospital outbreaks. Following cluster analysis based on amplification fragment length polymorphism (AFLP), these strains were grouped into three different clusters. The same clusters were obtained using protein profiles generated by MALDI-FTICR MS. Subsequent intact protein analysis by electrospray ionization (ESI)-collision-induced dissociation (CID)-FTICR MS was applied to identify protein isoforms that contribute to the separation of the different clusters, illustrating the additional advantage of this

  5. Effect of feeding isolates of anaerobic fungus Neocallimastix sp. CF 17 on growth rate and fibre digestion in buffalo calves.

    PubMed

    Paul, Shyam S; Deb, Sitangshu M; Punia, Balbir S; Das, Kalyan S; Singh, Ghansham; Ashar, Manisha N; Kumar, Rajiv

    2011-06-01

    In this investigation, the effects of feeding encapsulated cells (rhizomycelia and zoospores) of a fibrolytic isolate from an anaerobic fungus (Neocallimastix sp. CF 17) on nutrient digestion, ruminal fermentation, microbial populations, enzyme profile and growth performance were evaluated in buffaloes. In three in vitro studies, the true digestibility of wheat straw was increased after addition of CF 17 to buffalo rumen fluid (p < 0.05). In Exp. 1, three groups of six buffaloes each (initial BW [body weight] 148 +/- 12.0 kg) were allotted to three dosing regimes: Group 1 received 200 ml of liquid culture of Neocallimastix sp. CF 17 (about 10(6) TFU [thallus-forming units]/ml); Group 2 received an encapsulated culture of the same fungi prepared from 200 ml liquid culture; Group 3: received 200 ml of autoclaved culture (Control). The supplementations were given weekly for four weeks (on days 1,7, 14 and 21). During the dosing period, the average daily gain of Group 2 was higher than in the Control group (444 g/d compared with 264 g/d; p < 0.05). Furthermore, the digestibility of organic matter increased in Group 1 and 2 compared with the Control (64.8, 64.0 and 60.4% respectively; p < 0.05), resulting in an increase in the total digestible nutrient (TDN) percent of ration (p < 0.05). But these effects disappeared post-dosing. There were also an increase in concentration of volatile fatty acids, trichloroacetic acid precipitable N and number of fibrolytic microbes in the rumen during the dosing period (p < 0.05), but these effects declined post-dosing. Results of Exp 2., where the encapsulated culture was applied at intervals of 4 d or 8 d for 120 d, showed that a shorter dosing frequency did not improve growth performance or feed intake. However, independent of the dosing frequency the growth rate of both groups fed the encapsulated culture were about 20% higher than in the Control group (p < 0.05). The present study showed that encapsulated fungi have a high

  6. Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka.

    PubMed

    Honein, Karim; Jagoda, S S S De S; Arulkanthan, Appudurai; Ushio, Hideki; Asakawa, Shuichi

    2016-01-01

    Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka. PMID:27516512

  7. Draft Genome Sequences of Citrobacter freundii Strains CF04 and A41 Isolated from Moribund, Septicemic Giant Gourami (Osphronemus goramy) in Sri Lanka

    PubMed Central

    Jagoda, S. S. S. De S.; Arulkanthan, Appudurai; Ushio, Hideki

    2016-01-01

    Citrobacter freundii is a Gram-negative opportunistic pathogen associated with many infectious conditions including septicemia in humans and animals. Here, we announce the draft genome sequences of two multidrug-resistant C. freundii strains (CF04 and A41) isolated from septicemic giant gourami (Osphronemus goramy) collected from aquaria in Sri Lanka. PMID:27516512

  8. Neopetrosiquinones A and B, sesquiterpene benzoquinones isolated from the deep-water sponge Neopetrosia cf. proxima.

    PubMed

    Winder, Priscilla L; Baker, Heather L; Linley, Patricia; Guzmán, Esther A; Pomponi, Shirley A; Diaz, M Cristina; Reed, John K; Wright, Amy E

    2011-11-15

    Two new marine-derived sesquiterpene benzoquinones which we designate as neopetrosiquinones A (1) and B (2), have been isolated from a deep-water sponge of the family Petrosiidae. The structures were elucidated on the basis of their spectroscopic data. Compounds 1 and 2 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC(50) values of 3.7 and 9.8 μM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC(50) values of 6.1 and 13.8 μM, respectively. Neopetrosiquinone A (1) also inhibited the in vitro proliferation of the AsPC-1 human pancreatic carcinoma cell line with an IC(50) value of 6.1 μM. The compounds are structurally related to alisiaquinone A, cyclozonarone, and xestoquinone. PMID:22014756

  9. Neopetrosiquinones A and B, Sesquiterpene Benzoquinones Isolated from the Deep-water Sponge Neopetrosia cf. proxima

    PubMed Central

    Winder, Priscilla L.; Baker, Heather L.; Linley, Patricia; Guzmán, Esther; Pomponi, Shirley A.; Diaz, M. Cristina; Reed, John K.; Wright, Amy E.

    2011-01-01

    Two new marine-derived sesquiterpene benzoquinones which we designate as neopetrosiquinone A (1) and B (2), have been isolated from a deep-water sponge of the family Petrosiidae. The structures were elucidated on the basis of their spectroscopic data. Compounds 1 and 2 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC50 values of 3.7 and 9.8 μM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC50 values of 6.1 and 13.8 μM, respectively. Neopetrosiquinone A (1) also inhibited the in vitro proliferation of the AsPC-1 human pancreatic carcinoma cell line with an IC50 value of 6.1 μM. The compounds are structurally related to alisiaquinone A, cyclozonarone and xestoquinone. PMID:22014756

  10. Doripenem versus Pseudomonas aeruginosa In Vitro: Activity against Characterized Isolates, Mutants, and Transconjugants and Resistance Selection Potential

    PubMed Central

    Mushtaq, Shazad; Ge, Yigong; Livermore, David M.

    2004-01-01

    Doripenem is a broad-spectrum parenteral carbapenem under clinical development in Japan and North America. Its activities against (i) Pseudomonas aeruginosa isolates with graded levels of intrinsic efflux-type resistance, (ii) mutants with various combinations of AmpC and OprD expression, (iii) PU21 transconjugants with class A and D β-lactamases, and (iv) P. aeruginosa isolates with metallo-β-lactamases were tested by the agar dilution method of the National Committee for Clinical Laboratory Standards. Selection of resistant P. aeruginosa mutants was investigated in single- and multistep procedures. Doripenem MICs for isolates without acquired resistance mostly were 0.12 to 0.5 μg/ml, whereas meropenem MICs were 0.25 to 0.5 μg/ml and imipenem MICs were 1 to 2 μg/ml. The MICs of doripenem, meropenem, ertapenem, and noncarbapenems for isolates with increased efflux-type resistance were elevated, whereas the MICs of imipenem were less affected. The MICs of doripenem were increased by the loss of OprD but not by derepression of AmpC; nevertheless, and as with other carbapenems, the impermeability-determined resistance caused by the loss of OprD corequired AmpC activity and was lost in OprD− mutants also lacking AmpC. The TEM, PSE, PER, and OXA enzymes did not significantly protect P. aeruginosa PU21 against the activity of doripenem, whereas MICs of ≥16 μg/ml were seen for clinical isolates with VIM and IMP metallo-β-lactamases. Resistant mutants seemed to be harder to select with doripenem than with other carbapenems (or noncarbapenems), and the fold increases in the MICs were smaller for the resistant mutants. Single-step doripenem mutants were mostly resistant only to carbapenems and had lost OprD; multistep mutants had broader resistance, implying the presence of additional mechanisms, putatively including up-regulated efflux. Most mutants selected with aminoglycosides and quinolones had little or no cross-resistance to carbapenems, including doripenem

  11. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

    PubMed

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  12. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

    PubMed Central

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  13. Post-antibiotic effect of orbifloxacin against Escherichia coli and Pseudomonas aeruginosa isolates from dogs.

    PubMed

    Harada, Kazuki; Shimizu, Takae; Kataoka, Yasushi; Takahashi, Toshio

    2012-01-01

    Orbifloxacin is a fluoroquinolone drug used widely in companion animal medicine. In this study, we firstly determined post-antibiotic effects (PAEs) and post-antibiotic sub-minimum inhibitory concentrations (MIC) effects (PA-SMEs) of orbifloxacin for two strains each of Escherichia coli and Pseudomonas aeruginosa from dogs, and these parameters were compared with those of enrofloxacin. At twice the MIC, the PAEs of orbifloxacin ranged from -0.28-0.93 h (mean, 0.29 h) for E. coli and -0.18-1.18 h (mean, 0.37 h) for P. aeruginosa. These parameters were not significantly different for E. coli and shorter for P. aeruginosa, compared to enrofloxacin (P < 0.05). Continued exposure to 0.1, 0.2, and 0.3 the MIC of orbifloxacin resulted in average PA-SMEs of 0.55, 1.11, and 2.03 h, respectively, for E. coli, and 1.04, 1.40, and 2.47 h, respectively, for P. aeruginosa. These PA-SMEs, which had no significant differences with those of enrofloxacin, were significantly longer than the corresponding PAEs (P < 0.05). These results suggest that the PA-SME of orbifloxacin for E. coli and P. aeruginosa can be meaningfully prolonged by increase of sub-MICs. PMID:22433170

  14. Post-antibiotic effect of orbifloxacin against Escherichia coli and Pseudomonas aeruginosa isolates from dogs

    PubMed Central

    2012-01-01

    Orbifloxacin is a fluoroquinolone drug used widely in companion animal medicine. In this study, we firstly determined post-antibiotic effects (PAEs) and post-antibiotic sub-minimum inhibitory concentrations (MIC) effects (PA-SMEs) of orbifloxacin for two strains each of Escherichia coli and Pseudomonas aeruginosa from dogs, and these parameters were compared with those of enrofloxacin. At twice the MIC, the PAEs of orbifloxacin ranged from -0.28-0.93 h (mean, 0.29 h) for E. coli and -0.18-1.18 h (mean, 0.37 h) for P. aeruginosa. These parameters were not significantly different for E. coli and shorter for P. aeruginosa, compared to enrofloxacin (P < 0.05). Continued exposure to 0.1, 0.2, and 0.3 the MIC of orbifloxacin resulted in average PA-SMEs of 0.55, 1.11, and 2.03 h, respectively, for E. coli, and 1.04, 1.40, and 2.47 h, respectively, for P. aeruginosa. These PA-SMEs, which had no significant differences with those of enrofloxacin, were significantly longer than the corresponding PAEs (P < 0.05). These results suggest that the PA-SME of orbifloxacin for E. coli and P. aeruginosa can be meaningfully prolonged by increase of sub-MICs. PMID:22433170

  15. Five isolated pentagon rule isomers of higher fullerene C94 captured as chlorides and CF3 derivatives: C94(34)Cl14, C94(61)Cl20, C94(133)Cl22, C94(42)(CF3)16, and C94(43)(CF3)18.

    PubMed

    Tamm, Nadezhda B; Yang, Shangfeng; Wei, Tao; Troyanov, Sergey I

    2015-03-16

    High-temperature chlorination and trifluoromethylation of C94 isomeric mixtures followed by single-crystal X-ray diffraction with the use of synchrotron radiation resulted in the structure determination of C94(34)Cl14, C94(61)Cl20, C94(133)Cl22, C94(42)(CF3)16, and C94(43)(CF3)18. Their addition patterns are stabilized by the formation of isolated C═C bonds and aromatic substructures. Four cage isomers of C94 (nos. 34, 42, 43, and 133) have been experimentally confirmed for the first time. PMID:25698345

  16. 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

  17. blaVIM-2 Cassette-Containing Novel Integrons in Metallo-β-Lactamase-Producing Pseudomonas aeruginosa and Pseudomonas putida Isolates Disseminated in a Korean Hospital

    PubMed Central

    Lee, Kyungwon; Lim, Jong Back; Yum, Jong Hwa; Yong, Dongeun; Chong, Yunsop; Kim, June Myung; Livermore, David M.

    2002-01-01

    We investigated the phenotypic and genetic properties of metallo-β-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 β-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the blaVIM-2 genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had blaVIM located downstream of a variant of aacA4. blaVIM also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-β-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance. PMID:11897589

  18. Changing susceptibility of Pseudomonas aeruginosa isolates from cystic fibrosis patients with the clinical use of newer antibiotics.

    PubMed Central

    Bosso, J A; Allen, J E; Matsen, J M

    1989-01-01

    To detect a change in antibiotic susceptibility patterns in Pseudomonas aeruginosa isolates upon the introduction and clinical use of ciprofloxacin, aztreonam, and ceftazidime, MICs for clinical isolates collected before introduction of the antibiotics, during early clinical use, and later were determined for these and seven other antipseudomonal antibiotics. Concomitant resistance to two or more antibiotics was also studied. Over the three study periods, rates of susceptibility to 9 of the 10 antibiotics decreased. The largest decrease occurred with ceftazidime. Analysis of subsets of isolates from patients treated with ciprofloxacin or aztreonam also showed declining susceptibility to the latter but a stabilization of susceptibility to the former after an initial decline. Concomitant resistance within and among antibiotic classes was common. PMID:2499252

  19. In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.

    PubMed

    Fernández-Olmos, Ana; García-Castillo, María; Maiz, Luis; Lamas, Adelaida; Baquero, Fernando; Cantón, Rafael

    2012-08-01

    The ability of antibiotics used in bronchopulmonary infections in cystic fibrosis (CF) patients to prevent Pseudomonas aeruginosa early biofilm formation was studied using a biofilm microtitre assay with 57 non-mucoid P. aeruginosa isolates (44 first colonisers and 13 recovered during the initial intermittent colonisation stage) obtained from 35 CF patients. Minimum biofilm inhibitory concentrations (BICs) of levofloxacin, ciprofloxacin, imipenem, ceftazidime, tobramycin, colistin and azithromycin were determined by placing a peg lid with a formed biofilm onto microplates containing antibiotics. A modification of this protocol consisting of antibiotic challenge during biofilm formation was implemented in order to determine the biofilm prevention concentration (BPC), i.e. the minimum concentration able to prevent biofilm formation. The lowest BPCs were for fluoroquinolones, tobramycin and colistin and the highest for ceftazidime and imipenem. The former antibiotics had BPCs identical to or only slightly higher than their minimum inhibitory concentrations (MICs) determined by standard Clinical and Laboratory Standards Institute (CLSI) microdilution and were also active on formed biofilms as reflected by their low BIC values. In contrast, ceftazidime and imipenem were less effective for prevention of biofilm formation and on formed biofilms. In conclusion, the new BPC parameter determined in non-mucoid P. aeruginosa isolates recovered during early colonisation stages in CF patients supports early aggressive antimicrobial treatment guidelines in first P. aeruginosa-colonised CF patients. PMID:22727530

  20. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis.

    PubMed

    Clark, Shawn T; Diaz Caballero, Julio; Cheang, Mary; Coburn, Bryan; Wang, Pauline W; Donaldson, Sylva L; Zhang, Yu; Liu, Mingyao; Keshavjee, Shaf; Yau, Yvonne C W; Waters, Valerie J; Elizabeth Tullis, D; Guttman, David S; Hwang, David M

    2015-01-01

    Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to β-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample. PMID:26047320

  1. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis

    PubMed Central

    Clark, Shawn T.; Diaz Caballero, Julio; Cheang, Mary; Coburn, Bryan; Wang, Pauline W.; Donaldson, Sylva L.; Zhang, Yu; Liu, Mingyao; Keshavjee, Shaf; Yau, Yvonne C.W.; Waters, Valerie J.; Elizabeth Tullis, D.; Guttman, David S.; Hwang, David M.

    2015-01-01

    Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to β-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample. PMID:26047320

  2. Pseudomonas aeruginosa and Periodontal Pathogens in the Oral Cavity and Lungs of Cystic Fibrosis Patients: a Case-Control Study.

    PubMed

    Rivas Caldas, Rocio; Le Gall, Florence; Revert, Krista; Rault, Gilles; Virmaux, Michèle; Gouriou, Stephanie; Héry-Arnaud, Geneviève; Barbier, Georges; Boisramé, Sylvie

    2015-06-01

    Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. Lung destruction is the principal cause of death by chronic Pseudomonas aeruginosa colonization. There is a high prevalence of oropharyngeal anaerobic bacteria in sputum of CF patients. This study was carried out due to the lack of results comparing subgingival periodontal pathogenic bacteria between the oral cavity and lungs in patients with CF in relation with P. aeruginosa presence. Our first goal was to detect P. aeruginosa in oral and sputum samples by culture and molecular methods and to determine clonality of isolates. In addition, subgingival periodontal anaerobic bacteria were searched for in sputum. A cross-sectional pilot case-control study was conducted in the CF Reference Center in Roscoff, France. Ten CF patients with a ΔF508 homozygous mutation (5 chronically colonized [CC] and 5 not colonized [NC]) were enrolled. P. aeruginosa was detected in saliva, sputum, and subgingival plaque samples by real-time quantitative PCR (qPCR). Subsequently, periodontal bacteria were also detected and quantified in subgingival plaque and sputum samples by qPCR. In CC patients, P. aeruginosa was recovered in saliva and subgingival plaque samples. Sixteen P. aeruginosa strains were isolated in saliva and sputum from this group and compared by pulsed-field gel electrophoresis (PFGE). Subgingival periodontal anaerobic bacteria were found in sputum samples. A lower diversity of these species was recovered in the CC patients than in the NC patients. The presence of the same P. aeruginosa clonal types in saliva and sputum samples underlines that the oral cavity is a possible reservoir for lung infection. PMID:25854483

  3. [Protein a production in coagulase-negative or clumping factor (CF)-negative isolates of Staphylococcus aureus].

    PubMed

    Piechowicz, Lidia; Garbacz, Katarzyna; Wisniewska, Katarzyna; Dajnowska-Stanczewa, Agata; Galiński, Janusz

    2005-01-01

    The aim of study was to determine a production of proteinA in coagulase-negative Staphylococcus aureus (CNSA) or CF-negative S. aureus (CFNSA) strains. 59 CNSA and 18 CFNSA strains were isolated between 1997 and 2003 from different clinical specimens. The Protein A production was determined by immunoblotting method. The presence of protein A gene (spa) was investigated using the polymerase chain reaction (PCR). Two sets of phages and RFLP (Restriction Fragment Lenth Polymorphism) of coa gene method were used for typing strains. The results proved that the lack of ability of protein A production occurs more frequently in protein A-negative CFNSA strains with compare to the CNSA, which are protein A-positive for the majority of strains. Deficiencies of protein A, doesn't seem to be caused by the loss of spa gene. Protein A-negative CFNSA strains have phagotypes, RFLP and antibiotic resistant patterns which differ them from protein A-negative CNSA strains. Almost all of protein A-negative CFNSA and CNSA strains are resistant to methicillin. PMID:16494201

  4. Presence of blaPER-1 and blaVEB-1 beta-lactamase genes among isolates of Pseudomonas aeruginosa from South West of Iran.

    PubMed

    Davodian, Elham; Sadeghifard, Nourkhoda; Ghasemian, Abdolmajid; Noorbakhsh, Samileh

    2016-09-01

    Pseudomonas aeruginosa isolates have acquired resistance to antibiotics such as novel beta-lactams. The aim of this study was to investigate the blaPER-1, blaVEB-1, and blaPSE-1 genes among isolates of P. aeruginosa among intensive care unit (ICU) patients. Sixty-five isolates were collected. The antibiotic susceptibility testing and combined disk tests were performed to detect the isolates producing extended spectrum beta-lactamases (ESBLs) among ceftazidime-resistant isolates. Polymerase chain reaction (PCR) amplification of blaPER-1, blaVEB-1, and blaPSE-1 genes was conducted. Ten (15.3%) isolates were ESBL-positive, of which 40% (n=4) belonged to males and 60% (n=6) were collected from females. Moreover, two and one isolates harbored blaPER-1 and blaVEB-1 genes, respectively. PMID:26944896

  5. Comparison of susceptibility of cystic-fibrosis-related and non-cystic-fibrosis-related Pseudomonas aeruginosa to chlorine-based disinfecting solutions: implications for infection prevention and ward disinfection.

    PubMed

    Moore, John E; Rendall, Jacqueline C

    2014-09-01

    Multidrug-resistant (MDR) Pseudomonas aeruginosa isolated from cystic fibrosis (CF) sputum was shown to be more tolerant to the most commonly used chlorine-based disinfecting agent in the UK, with approximately 7 out of 10 isolates surviving a residual free chlorine (RFC) concentration of 500 p.p.m., when compared with antibiotic-sensitive invasive P. aeruginosa from a non-CF blood culture source, where 8 out of 10 isolates were killed at a RFC concentration of 100 p.p.m. All CF isolates were killed at 1000 p.p.m. chlorine. Additional studies were performed to examine factors that influenced the concentration of RFC from chlorine-based (sodium dichloroisocyanurate) disinfecting agents in contact with CF sputum and their components (bacterial cells, glycocalyx) to assess the reduction of the bactericidal activity of such disinfecting agents. Pseudomonas glycocalyx had a greater inhibitory effect of chlorine deactivation than bacterial cells. Calibration curves demonstrated the relative deactivating capacity on RFC from clinical soils, in the order pus>CF sputum>wound discharge fluid/synovial fluid>ascites fluid>bile, where quantitatively each 1 % (w/v) CF sputum reduced the RFC by 43 p.p.m. Sublethal stressing of P. aeruginosa with chlorine resulted in lowered susceptibility to colistin (P = 0.0326) but not to meropenem, tobramycin or ciprofloxacin. In conclusion, heavy contamination of healthcare fomites with CF sputum containing MDR P. aeruginosa may result in exhaustion of RFC, and this, combined with an increased resistance to chlorine with such strains, may lead to their survival and increased antibiotic resistance in such environments. CF infection prevention strategies in such scenarios should therefore target interventions with increased concentrations of chlorine to ensure the eradication of MDR P. aeruginosa from the CF healthcare environment. PMID:24925907

  6. Pseudomonas aeruginosa Phenotypes Associated With Eradication Failure in Children With Cystic Fibrosis

    PubMed Central

    Mayer-Hamblett, Nicole; Ramsey, Bonnie W.; Kulasekara, Hemantha D.; Wolter, Daniel J.; Houston, Laura S.; Pope, Christopher E.; Kulasekara, Bridget R.; Armbruster, Catherine R.; Burns, Jane L.; Retsch-Bogart, George; Rosenfeld, Margaret; Gibson, Ronald L.; Miller, Samuel I.; Khan, Umer; Hoffman, Lucas R.

    2014-01-01

    Background. Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized. Methods. We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation. Results. Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03–3.83] and 2.14 [1.32–3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial. Conclusions. Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing. PMID:24863401

  7. 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

  8. Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

    PubMed Central

    Marvig, Rasmus Lykke; Damkiær, Søren; Khademi, S. M. Hossein; Markussen, Trine M.; Molin, Søren

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. PMID:24803516

  9. Isolation and characterization of a Pseudomonas aeruginosa from a virgin Brazilian Amazon region with potential to degrade atrazine.

    PubMed

    Fernandes, Ana Flavia Tonelli; da Silva, Michelle Barbosa Partata; Martins, Vinicius Vicente; Miranda, Carlos Eduardo Saraiva; Stehling, Eliana Guedes

    2014-12-01

    The use of pesticides to increase agricultural production can result in the contamination of the environment, causing changes in the genetic structure of organisms and in the loss of biodiversity. This practice is also inducing changes in the rainforest ecosystem. In this work, a Pseudomonas aeruginosa isolated from a preservation soil area of the Brazilian Amazon Forest, without usage of any pesticide, was evaluated for its potential to degrade atrazine. This isolate presented all responsible genes (atzA, atzB, atzC, atzD, atzE, and atzF) for atrazine mineralization and demonstrated capacity to use atrazine as a nitrogen source, having achieved a reduction of 44 % of the initial concentration of atrazine after 24 h. These results confirm gene dispersion and/or a possible contamination of the area with the herbicide, which reinforces global concern of the increase and intensive use of pesticides worldwide. PMID:25035056

  10. 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

  11. In71, an Enterobacter cloacae blaVIM-1-carrying integron related to In70.2 from Italian Pseudomonas aeruginosa isolates: a SENTRY Antimicrobial Surveillance Program report.

    PubMed

    Castanheira, Mariana; Sader, Hélio S; Jones, Ronald N; Debbia, Eugenio; Picão, Renata C; Gales, Ana C

    2007-01-01

    An Enterobacter cloacae strain showing decreased susceptibility to carbapenems was isolated from a blood culture of a patient hospitalized in Genoa, Italy, and screened for the presence of metallo-beta-lactamase (MBL) genes as part of the SENTRY Antimicrobial Surveillance Program. A bla(VIM-1)-carrying integron named In71 nearly identical to In70.2 reported in Pseudomonas aeruginosa strains isolated from various Italian cities since 2001 was identified in this strain. Interestingly, the In71 did not carry aadA1 nor possess the ISPa7 usually found in the P. aeruginosa integron In70.2. Mobilization of MBL genes from P. aeruginosa to members of the Enterobacteriaceae family is very worrisome because the rapid and wide dissemination of these potent antimicrobial resistance mechanisms could jeopardize the clinical use of carbapenems for the treatment of multidrug-resistant Enterobacteriaceae infections. PMID:17650966

  12. Iron Depletion Enhances Production of Antimicrobials by Pseudomonas aeruginosa

    PubMed Central

    Nguyen, Angela T.; Jones, Jace W.; Ruge, Max A.; Kane, Maureen A.

    2015-01-01

    ABSTRACT Cystic fibrosis (CF) is a heritable disease characterized by chronic, polymicrobial lung infections. While Staphylococcus aureus is the dominant lung pathogen in young CF patients, Pseudomonas aeruginosa becomes predominant by adulthood. P. aeruginosa produces a variety of antimicrobials that likely contribute to this shift in microbial populations. In particular, secretion of 2-alkyl-4(1H)-quinolones (AQs) contributes to lysis of S. aureus in coculture, providing an iron source to P. aeruginosa both in vitro and in vivo. We previously showed that production of one such AQ, the Pseudomonas quinolone signal (PQS), is enhanced by iron depletion and that this induction is dependent upon the iron-responsive PrrF small RNAs (sRNAs). Here, we demonstrate that antimicrobial activity against S. aureus during coculture is also enhanced by iron depletion, and we provide evidence that multiple AQs contribute to this activity. Strikingly, a P. aeruginosa ΔprrF mutant, which produces very little PQS in monoculture, was capable of mediating iron-regulated growth suppression of S. aureus. We show that the presence of S. aureus suppresses the ΔprrF1,2 mutant's defect in iron-regulated PQS production, indicating that a PrrF-independent iron regulatory pathway mediates AQ production in coculture. We further demonstrate that iron-regulated antimicrobial production is conserved in multiple P. aeruginosa strains, including clinical isolates from CF patients. These results demonstrate that iron plays a central role in modulating interactions of P. aeruginosa with S. aureus. Moreover, our studies suggest that established iron regulatory pathways of these pathogens are significantly altered during polymicrobial infections. IMPORTANCE Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are a significant cause of morbidity and mortality, as the interplay between these two organisms exacerbates infection. This is in part due to enhanced

  13. Isolation, identification, and algicidal activity of aerobic denitrifying bacterium R11 and its effect on Microcystis aeruginosa.

    PubMed

    Su, Jun-Feng; Shao, Si-Cheng; Huang, Ting-Lin; Ma, Fang; Zhang, Kai; Wen, Gang; Zheng, Sheng-Chen

    2016-01-01

    Recently, algicidal bacteria have attracted attention as possible agents for the inhibition of algal water blooms. In this study, an aerobic denitrifying bacterium, R11, with high algicidal activity against the toxic Microcystis aeruginosa was isolated from lake sediments. Based on its physiological characteristics and 16S rRNA gene sequence, it was identified as Raoultella, indicating that the bacterium R11 has a good denitrifying ability at 30 °C and can reduce the concentration of nitrate-N completely within 36 h. Additionally, different algicidal characteristics against Microcystis aeruginosa were tested. The results showed that the initial bacterial cell density and algal cell densities strongly influence the removal rates of chlorophyll a. Algicidal activity increased with an increase in the bacterial cell density. With densities of bacterial culture at over 2.4 × 10(5) cell/mL, algicidal activity of up to 80% was obtained in 4 days. We have demonstrated that, with the low initial algal cell density (OD680 less than 0.220), the algicidal activity reached was higher than 90% after 6 days. PMID:27232395

  14. Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa

    PubMed Central

    Scott, Martin; Worden, Paul; Huntington, Peter; Hudson, Bernard; Karagiannis, Thomas; Charles, Ian G.; Djordjevic, Steven P.

    2016-01-01

    Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a blaGES-5–aacA4–gcuE15–aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa. PMID:26962050

  15. Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa.

    PubMed

    Roy Chowdhury, Piklu; Scott, Martin; Worden, Paul; Huntington, Peter; Hudson, Bernard; Karagiannis, Thomas; Charles, Ian G; Djordjevic, Steven P

    2016-03-01

    Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a blaGES-5-aacA4-gcuE15-aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa. PMID:26962050

  16. Similar Frequencies of Pseudomonas aeruginosa Isolates Producing KPC and VIM Carbapenemases in Diverse Genetic Clones at Tertiary-Care Hospitals in Medellín, Colombia

    PubMed Central

    Vanegas, Johanna M.; Cienfuegos, Astrid V.; Ocampo, Ana M.; López, Lucelly; del Corral, Helena; Roncancio, Gustavo; Sierra, Patricia; Echeverri-Toro, Lina; Ospina, Sigifredo; Maldonado, Natalia; Robledo, Carlos; Restrepo, Andrea

    2014-01-01

    Carbapenem-resistant Pseudomonas aeruginosa has become a serious health threat worldwide due to the limited options available for its treatment. Understanding its epidemiology contributes to the control of antibiotic resistance. The aim of this study was to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant P. aeruginosa isolates in five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to March 2014. All hospitalized patients infected by carbapenem-resistant P. aeruginosa were included. Clinical information was obtained from medical records. Molecular analyses included PCR for detection of blaVIM, blaIMP, blaNDM, blaOXA-48, and blaKPC genes plus pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. A total of 235 patients were enrolled: 91.1% of them were adults (n = 214), 88.1% (n = 207) had prior antibiotic use, and 14.9% (n = 35) had urinary tract infections. The blaVIM-2 and blaKPC-2 genes were detected in 13.6% (n = 32) and 11.5% (n = 27), respectively, of all isolates. Two isolates harbored both genes simultaneously. For KPC-producing isolates, PFGE revealed closely related strains within each hospital, and sequence types (STs) ST362 and ST235 and two new STs were found by MLST. With PFGE, VIM-producing isolates appeared highly diverse, and MLST revealed ST111 in four hospitals and five new STs. These results show that KPC-producing P. aeruginosa is currently disseminating rapidly and occurring at a frequency similar to that of VIM-producing P. aeruginosa isolates (approximately 1:1 ratio) in Medellín, Colombia. Diverse genetic backgrounds among resistant strains suggest an excessive antibiotic pressure resulting in the selection of resistant strains. PMID:25210071

  17. Similar frequencies of Pseudomonas aeruginosa isolates producing KPC and VIM carbapenemases in diverse genetic clones at tertiary-care hospitals in Medellín, Colombia.

    PubMed

    Vanegas, Johanna M; Cienfuegos, Astrid V; Ocampo, Ana M; López, Lucelly; del Corral, Helena; Roncancio, Gustavo; Sierra, Patricia; Echeverri-Toro, Lina; Ospina, Sigifredo; Maldonado, Natalia; Robledo, Carlos; Restrepo, Andrea; Jiménez, J Natalia

    2014-11-01

    Carbapenem-resistant Pseudomonas aeruginosa has become a serious health threat worldwide due to the limited options available for its treatment. Understanding its epidemiology contributes to the control of antibiotic resistance. The aim of this study was to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant P. aeruginosa isolates in five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to March 2014. All hospitalized patients infected by carbapenem-resistant P. aeruginosa were included. Clinical information was obtained from medical records. Molecular analyses included PCR for detection of bla(VIM), bla(IMP), bla(NDM), bla(OXA-48), and bla(KPC) genes plus pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. A total of 235 patients were enrolled: 91.1% of them were adults (n = 214), 88.1% (n = 207) had prior antibiotic use, and 14.9% (n = 35) had urinary tract infections. The bla(VIM-2) and bla(KPC-2) genes were detected in 13.6% (n = 32) and 11.5% (n = 27), respectively, of all isolates. Two isolates harbored both genes simultaneously. For KPC-producing isolates, PFGE revealed closely related strains within each hospital, and sequence types (STs) ST362 and ST235 and two new STs were found by MLST. With PFGE, VIM-producing isolates appeared highly diverse, and MLST revealed ST111 in four hospitals and five new STs. These results show that KPC-producing P. aeruginosa is currently disseminating rapidly and occurring at a frequency similar to that of VIM-producing P. aeruginosa isolates (approximately 1:1 ratio) in Medellín, Colombia. Diverse genetic backgrounds among resistant strains suggest an excessive antibiotic pressure resulting in the selection of resistant strains. PMID:25210071

  18. Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates.

    PubMed

    Thrane, Sandra Wingaard; Taylor, Véronique L; Lund, Ole; Lam, Joseph S; Jelsbak, Lars

    2016-07-01

    Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data. PMID:27098958

  19. Hypermutable Pseudomonas aeruginosa in Cystic fibrosis patients from two Brazilian cities.

    PubMed

    Lutz, Larissa; Leão, Robson Souza; Ferreira, Alex Guerra; Pereira, Dariane Castro; Raupp, Caroline; Pitt, Tyrone; Marques, Elizabeth Andrade; Barth, Afonso Luis

    2013-03-01

    Hypermutable (HPM) strains of Pseudomonas aeruginosa have been found at high frequencies in cystic fibrosis (CF) patients in Europe. We report the results of testing for HPM frequencies, mutator genotype, and antimicrobial resistance of P. aeruginosa strains from Brazilian CF patients. A modified disk diffusion technique was used to quantify antibiotic-resistant subpopulations of an isolate, and estimations of the frequency of mutation to rifampin resistance were determined for 705 isolates from 149 patients attending clinics in two Brazilian cities. Mutations in the mutS gene were detected by sequencing assays. We found 194 (27.5%) HPM isolates in samples from 99 (66.4%) patients. Thirty-five HPM isolates (18.0%) from 31 (31.3%) patients exhibited a high increased spontaneous mutation rate compared with controls, and eight isolates from six patients displayed a defective mutS gene. The dominant HPM population was associated with very low antibiotic resistance levels, while HPM subpopulations were generally more resistant to antimicrobials. A relatively high prevalence of HPM P. aeruginosa in CF patients was associated with surprisingly low antibiotic resistance levels, in contrast to some earlier studies. PMID:23303495

  20. Isolation, purification, and characterization of the major autolysin from Pseudomonas aeruginosa.

    PubMed

    Watt, S R; Clarke, A J

    1997-11-01

    The major (26 kDa) autolysin from Pseudomonas aeruginosa was purified to apparent homogeneity by a combination of preparative electrophoresis, ion-exchange, and dye-ligand chromatographies. This purification was facilitated by the development of a spot-assay that involved the spotting and subsequent incubation of autolysin samples on polyacrylamide gels containing peptidoglycan. The pl of the 26-kDa autolysin was determined to be between 3.5 and 4 and disulfide bonds within the enzyme were essential for activity. The autolysin catalyzed the release of reducing sugars from the peptidoglycans of Pseudomonas aeruginosa and Escherichia coli indicating it to be a beta-glycosidase. It was ineffective at hydrolysing the peptidoglycan from Gram-positive bacteria and the O-acetylated peptidoglycans from either Proteus mirabilis or Staphylococcus aureus. The N-terminal sequence of the purified autolysin was determined to be His-Glu-Pro-Pro-Gly. The 26-kDa autolysin together with a 29-kDa autolysin was determined to be secreted into the medium by a mechanism that involves the production and release of surface membrane vesicles during normal growth, but the enzymes were not found free and active in culture broth supernatants. PMID:9436306

  1. Genes Required for and Effects of Alginate Overproduction Induced by Growth of Pseudomonas aeruginosa on Pseudomonas Isolation Agar Supplemented with Ammonium Metavanadate

    PubMed Central

    Damron, F. Heath; Barbier, Mariette; McKenney, Elizabeth S.; Schurr, Michael J.

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, by transcriptomics, and in a murine acute virulence model. The PA14 nonredundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan remodeling, uptake of phosphate and iron, phenazine biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa cultures growing in the presence of vanadate showed differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa, but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study. PMID:23794622

  2. Genetic analyses of Pseudomonas aeruginosa isolated from healthy captive snakes: evidence of high inter- and intrasite dissemination and occurrence of antibiotic resistance genes.

    PubMed

    Colinon, Céline; Jocktane, Dominique; Brothier, Elisabeth; Rossolini, Gian Maria; Cournoyer, Benoit; Nazaret, Sylvie

    2010-03-01

    Faecal carriage of Pseudomonas aeruginosa was investigated by selective plating and PCR identification test, among healthy captive snakes from zoological and private collections from France as well as from wild snakes from Guinea. P. aeruginosa faecal carriage among captive snakes was high (72 out of 83 individuals), but low among wild specimen (3 out of 23 individuals). Genetic diversity analyses of the isolates, based on SpeI-PFGE profiles, evidenced five dominant clones or clonal complexes spreading among snakes within a site and between sites and persisting over time. Similar clones or clonal complexes were detected from mouth swabs of the owners and from water and preys used to feed the snakes, evidencing various sources of snake colonization and the first cases of P. aeruginosa cross-contamination between snakes and owners. These observations led to the conclusion that P. aeruginosa behaves as an opportunistic species within snakes in captivity and that colonization and dissemination occurs consecutively to processes similar to those identified within the hospital. Antibiotic susceptibility testing showed that most isolates had a wild-type resistance profile except for one persistent clone isolated from both snakes and preys that harboured multiple antimicrobial resistance genes mediated by an integron carrying the qacH, aadB, aadA2 and cmlA10 cassettes, and a tetA(C)-carrying transposon. Biocides or antibiotics used in the zoological garden could have led to the acquisition of this integron. PMID:20002132

  3. New Isolated-Pentagon-Rule Isomer of C92 Isolated as Trifluoromethyl and Chlorido Derivatives: C92(38)(CF3)14/16 and C92(38)Cl20/22.

    PubMed

    Tamm, Nadezhda B; Troyanov, Sergey I

    2015-11-16

    High-temperature trifluoromethylation and chlorination of the C92 fraction followed by single-crystal X-ray diffraction with the use of synchrotron radiation resulted in the structure determination of C92(38)(CF3)14, five isomers of C92(38)(CF3)16, and C92(38)Cl20/22. Their addition patterns are stabilized by the formation of isolated C═C bonds and aromatic substructures. According to quantum-chemical calculations, the newly detected C1-C92(38) belongs to the most stable isomers of C92. PMID:26530220

  4. Mutations in NalC induce MexAB-OprM overexpression resulting in high level of aztreonam resistance in environmental isolates of Pseudomonas aeruginosa.

    PubMed

    Braz, Vânia S; Furlan, João Pedro R; Fernandes, Ana Flavia T; Stehling, Eliana G

    2016-08-01

    Pseudomonas aeruginosa is an opportunistic pathogen with high resistance to a wide variety of antimicrobials. The multidrug resistance pump MexAB-OprM promotes the efflux of various antibiotics, mostly when mutations accumulate in the transcriptional regulators MexR, NalC and NalD, thereby causing MexAB-OprM overexpression. In this work, a characterization of 50 P. aeruginosa isolates obtained from Brazilian agricultural soils to determine the reasons of their resistance to aztreonam was done. The majority of the isolates showed higher aztreonam resistance than wild-type strain by MIC method. DNA sequence analysis of mexR, nalC and nalD genes from 13 of these isolates showed the amino acid substitution in NalC for all tested isolates, just one mutation was detected in MexR and none in NalD. Furthermore, an increase in the level of mexA expression by real-time RT-PCR analysis in eight isolates harboring mutations in NalC was found. Although there was not a relationship between MIC of aztreonam and the level of mexA expression, on the other hand, the results presented here suggest that novel mutations in NalC, including Arg97-Gly and Ala186-Thr, are related to MexAB-OprM overexpression causing aztreonam resistance in P. aeruginosa environmental isolates. PMID:27412168

  5. Metallo-beta-Lactamase VIM-1, SPM-1, and IMP-1 Genes Among Clinical Pseudomonas aeruginosa Species Isolated in Zahedan, Iran

    PubMed Central

    Ghamgosha, Mehdi; Shahrekizahedani, Shahram; Kafilzadeh, Farshid; Bameri, Zakaria; Taheri, Ramezan Ali; Farnoosh, Gholamreza

    2015-01-01

    Background: One of the major clinical problems regarding Pseudomonas aeruginosa is attributed to metallo-beta-lactamases (MBL). This group of enzymes is a subset of beta lactamases which belong to group B of Ambler classification and cause hydrolysis of carbapenems. Based on epidemiological studies conducted worldwide, it is proved that prevalence of genes coding MBLs in P. aeruginosa species are different in various geographic zones and even in various hospitals. Therefore, according to the clinical importance of organisms generating MBLs, it is necessary to identify and control these bacteria in hospitals for therapeutic purposes. Objectives: The current study aimed to investigate the Metallo-beta-Lactamase VIM-1, SPM-1, and IMP-1 genes among clinical P. aeruginosa species isolated in Zahedan, Iran. Materials and Methods: The current study investigated the presence of MBL through phenotypic and genotypic methods and also the pattern of antibiotic resistance in P. aeruginosa species isolated in hospitals. The Minimum Inhibitory Concentration (MIC) against imipeneme was measured for 191 P. aeruginosa species isolated from Zahedan hospitals after identification through biochemical methods and determination of the antibiotic resistance pattern. Strains with MIC > 4 µg/mL were studied by phenotypic and genotypic methods. Results: The rate of resistance against imipeneme was 5.7% and after carrying out the phenotypic experiments, nine species were identified as of MBL producer. Seven species were confirmed by Polymerase Chain Reaction (PCR) method. Gene VIM-1 was the predominant gene among the positive (antibiotic resistant) species. Conclusions: The study results showed that MBL genes were present in some of the species isolated from Zahedan hospitals. Regarding the importance of MBL producer bacteria in hospitals, quick identification and evaluation of these clinical species can be considered as an important and basic step for treatment and control of pseudomonad

  6. Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

    PubMed Central

    Ieranò, Teresa; Lorè, Nicola Ivan; Bianconi, Irene; Silipo, Alba; Cozzolino, Flora; Lanzetta, Rosa; Molinaro, Antonio; Bernardini, Maria Lina; Bragonzi, Alessandra

    2009-01-01

    Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF. PMID:20037649

  7. 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

  8. Co-Carriage of blaKPC-2 and blaNDM-1 in Clinical Isolates of Pseudomonas aeruginosa Associated with Hospital Infections from India

    PubMed Central

    Paul, Deepjyoti; Dhar Chanda, Debadatta; Maurya, Anand Prakash; Mishra, Shweta; Chakravarty, Atanu; Sharma, Gauri Dutt; Bhattacharjee, Amitabha

    2015-01-01

    Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of blaKPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012–2013. The presence of blaKPC was confirmed by genotypic characterization followed by sequencing. Cloning of the blaKPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor blaKPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring blaNDM-1. The first detection of this integron mediated blaKPC-2 coexisting with blaNDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated blaKPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community. PMID:26714034

  9. In Vitro Susceptibility of Global Surveillance Isolates of Pseudomonas aeruginosa to Ceftazidime-Avibactam (INFORM 2012 to 2014).

    PubMed

    Nichols, Wright W; de Jonge, Boudewijn L M; Kazmierczak, Krystyna M; Karlowsky, James A; Sahm, Daniel F

    2016-08-01

    Broth microdilution antimicrobial susceptibility testing was performed for ceftazidime-avibactam and comparator agents against 7,062 clinical isolates of Pseudomonas aeruginosa collected from 2012 to 2014 in four geographic regions (Europe, Asia/South Pacific, Latin America, Middle East/Africa) as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance program. The majority of isolates were susceptible to ceftazidime-avibactam, with the proportions susceptible differing marginally across the four regions (MIC90, 8 to 16 μg/ml; 88.7 to 93.2% susceptible), in contrast to lower susceptibilities to the following comparator β-lactam agents: ceftazidime (MIC90, 32 to 64 μg/ml; 71.5 to 80.8% susceptible), meropenem (MIC90, >8 μg/ml; 64.9 to 77.4% susceptible), and piperacillin-tazobactam (MIC90, >128 μg/ml; 62.3 to 71.3% susceptible). Compared to the overall population, susceptibility to ceftazidime-avibactam of isolates that were nonsusceptible to ceftazidime (n = 1,627) was reduced to between 56.8% (Middle East/Africa; MIC90, 64 μg/ml) and 68.9% (Asia/South Pacific; MIC90, 128 μg/ml), but these percentages were higher than susceptibilities to other β-lactam agents (0 to 44% susceptible, depending on region and agent; meropenem MIC90, >8 μg/ml; 26.5 to 43.9% susceptible). For this subset of isolates, susceptibilities to amikacin (MIC90, >32 μg/ml; 53.2 to 80.0% susceptible) and colistin (MIC90, 1 μg/ml; 98.5 to 99.5% susceptible) were comparable to or higher than that of ceftazidime-avibactam. A similar observation was made with isolates that were nonsusceptible to meropenem (n = 1,926), with susceptibility to ceftazidime-avibactam between 67.8% (Middle East/Africa; MIC90, 64 μg/ml) and 74.2% (Europe; MIC90, 32 μg/ml) but again with reduced susceptibility to comparators except for amikacin (MIC90, >32 μg/ml; 56.8 to 78.7% susceptible) and colistin (MIC90, 1 μg/ml; 98.9 to 99.3% susceptible). Of the 8% of isolates

  10. 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

  11. Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection.

    PubMed

    Darch, Sophie E; McNally, Alan; Harrison, Freya; Corander, Jukka; Barr, Helen L; Paszkiewicz, Konrad; Holden, Stephen; Fogarty, Andrew; Crusz, Shanika A; Diggle, Stephen P

    2015-01-01

    The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF. PMID:25578031

  12. Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection

    PubMed Central

    Darch, Sophie E.; McNally, Alan; Harrison, Freya; Corander, Jukka; Barr, Helen L.; Paszkiewicz, Konrad; Holden, Stephen; Fogarty, Andrew; Crusz, Shanika A.; Diggle, Stephen P.

    2015-01-01

    The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF. PMID:25578031

  13. Hospital Isolates of Serratia marcescens Transferring Ampicillin, Carbenicillin, and Gentamicin Resistance to Other Gram-Negative Bacteria Including Pseudomonas aeruginosa

    PubMed Central

    Olexy, Vera M.; Bird, Thomas J.; Grieble, Hans G.; Farrand, Stephen K.

    1979-01-01

    Thirteen independent isolates of Serratia marcescens associated with nosocomial urinary tract infections were obtained from the clinical microbiology laboratory at Hines Veterans Administration Hospital. The isolates were resistant to at least ampicillin, carbenicillin, gentamicin, and tobramycin. They could be divided into two groups on the basis of their antibiotypes. Group I (9 strains) showed resistance to 13 antibiotics, including 3 beta-lactams, 6 aminoglycosides, tetracycline, sulfonamide, trimethoprim, and polymyxin B. Group II (4 strains) was resistant to 11 antibiotics, including 3 beta-lactams, 5 aminoglycosides, sulfonamide, trimethoprim, and polymyxin B. Donors from both groups transferred resistance traits to Escherichia coli. Transconjugants from matings with group II donors all acquired resistance to nine antibiotics, including the three beta-lactams, five aminoglycosides, and sulfonamide. Transconjugants from matings with group I donors were of varied antibiotypes, inheriting resistance to up to 11 of the 13 antibiotics. Resistances to trimethoprim and polymyxin B were never observed to transfer. E. coli transconjugants of each group were capable of transferring multiple-antibiotic resistance to several other members of the family Enterobacteriaceae. All group II S. marcescens and E. coli donors and all group I S. marcescens donors transferred carbenicillin, streptomycin, kanamycin, gentamicin, tobramycin, and sisomicin resistance to Pseudomonas aeruginosa. The results suggest that these S. marcescens strains harbor R factors of a broader host range than previously reported. PMID:106772

  14. Antibiotic resistance profiles of Pseudomonas aeruginosa isolated from various Greek aquatic environments.

    PubMed

    Olga, Pappa; Apostolos, Vantarakis; Alexis, Galanis; George, Vantarakis; Athena, Mavridou

    2016-05-01

    A large number of antibiotic-resistantP. aeruginosaisolates are continuously discharged into natural water basins mainly through sewage. However, the environmental reservoirs of antibiotic resistance factors are poorly understood. In this study, the antibiotic resistance patterns of 245 isolates from various aquatic sites in Greece were analysed. Twenty-three isolates with resistance patterns cefotaxime-aztreonam-ceftazidime, cefotaxime-aztreonam-meropenem, cefotaxime-ceftazidime-meropenem, cefotaxime-ceftazidime-aztreonam-meropenem and cefotaxime-ceftazidime-cefepime-aztreonam-meropenem were screened phenotypically for the presence of extended spectrum β-lactamases (ESBLs), while 77 isolates with various resistant phenotypes were screened for the presence of class 1 and class 2 integrase genes. The aztreonam-resistant isolates and ESBL producers were the main resistant phenotypes in all habitats tested. In 13/77 isolates class 1 integron was detected, while all tested isolates were negative for the presence of the class 2 integrase gene. CTX-M group 9 β-lactamase was present in a small number of isolates (three isolates) highlighting the emergence of ESBL genes in aquatic environments. As a conclusion, it seems that Greek water bodies could serve as a potential reservoir of resistantP. aeruginosaisolates posing threats to human and animal health. PMID:26917780

  15. Prevalence of Extended-Spectrum and Metallo β-Lactamase Production in AmpC β-Lactamase Producing Pseudomonas aeruginosa Isolates From Burns

    PubMed Central

    Rafiee, Roya; Eftekhar, Fereshteh; Tabatabaei, Seyyed Ahmad; Minaee Tehrani, Dariush

    2014-01-01

    Background: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Resistance of P. aeruginosa to β-lactam antibiotics may be the result of acquired resistance through mutation and over production of various antibiotic inactivating enzymes. This research aimed to determine the prevalence of extended-spectrum β-lactamases (ESBL) and metallo β-lactamase (MBL) production as well as the presence of their related genes among AmpC β-lactamase producing P. aeruginosa isolated from burns. Objectives: The current study aimed to determine the prevalence of class A ESBL and MBL production in relation to the presence of their related genes among AmpC β-lactamase producing P. aeruginosa isolated from burns. Materials and Methods: The antimicrobial susceptibility of 51 P. aeruginosa isolates from patients with burns was examined against 13 antibiotics by the disc diffusion method. Minimum inhibitory concentrations (MIC) for imipenem and ceftazidime were measured by the microdilution method. AmpC production was detected by AmpC disc and the modified three-dimensional extract tests. ESBL phenotype was confirmed by the double disc synergy test (DDST). Presence of β-lactamase genes was detected by specific primers and polymerase chain reaction (PCR). Results: All isolates were multidrug resistant. AmpC, ESBL and MBL production were observed in 35 (68.6%), 20 (39.2%) and 19 (37.3%) isolates, respectively. Overall, 43 isolates (84.3%) carried β-lactamase genes, out of which 31 (60.8%) harbored blaAmpC, 20 (39.2%) had blaTEM and 11 (21.6%) carried blaPER-1 genes. Among the AmpC producers, two isolates (6.5%) carried blaAmpC + blaESBL, 13 (41.9%) had blaAmpC + blaMBL and six (19.4%) produced the three enzymes. Conclusions: A high prevalence of multiple β-lactamase production was observed among the AmpC producers (60%), of which the majority co-produced AmpC and MBL. The current study results showed correlation between β-lactamase production and the

  16. 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

  17. 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

  18. Synthesis and characterization of Pseudomonas aeruginosa alginate-tetanus toxoid conjugate.

    PubMed

    Kashef, Nasim; Behzadian-Nejad, Qorban; Najar-Peerayeh, Shahin; Mousavi-Hosseini, Kamran; Moazzeni, Mohammad; Djavid, Gholamreza Esmaeeli

    2006-10-01

    Chronic infection with Pseudomonas aeruginosa is the main proven perpetrator of lung function decline and ultimate mortality in cystic fibrosis (CF) patients. Mucoid strains of this bacterium elaborate mucoid exopolysaccharide, also referred to as alginate. Alginate-based immunization of naïve animals elicits opsonic antibodies and leads to clearance of mucoid P. aeruginosa from the lungs. Alginate was isolated from mucoid P. aeruginosa strain 8821M by repeated ethanol precipitation, dialysis, proteinase and nuclease digestion, and chromatography. To improve immunogenicity, the purified antigen was coupled to tetanus toxoid (TT) with adipic acid dihydrazide (ADH) as a spacer and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as a linker. The reaction mixture was passed through a Sepharose CL-4B column. The resulting conjugate was composed of TT and large-size alginate polymer at a ratio of about 3 : 1; it was non-toxic and non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoid P. aeruginosa in the CF host. PMID:17005795

  19. Extensively Drug-Resistant Pseudomonas aeruginosa Isolates Containing blaVIM-2 and Elements of Salmonella Genomic Island 2: a New Genetic Resistance Determinant in Northeast Ohio

    PubMed Central

    Perez, Federico; Hujer, Andrea M.; Marshall, Steven H.; Ray, Amy J.; Rather, Philip N.; Suwantarat, Nuntra; Dumford, Donald; O'Shea, Patrick; Domitrovic, T. Nicholas J.; Salata, Robert A.; Chavda, Kalyan D.; Chen, Liang; Kreiswirth, Barry N.; Vila, Alejandro J.; Haussler, Susanne; Jacobs, Michael R.

    2014-01-01

    Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-β-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and β-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this region's health care system. PMID:25070102

  20. Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

    PubMed Central

    Cavalcanti, Felipe Lira de Sá; Mirones, Cristina Rodríguez; Paucar, Elena Román; Montes, Laura Álvarez; Leal-Balbino, Tereza Cristina; de Morais, Marcia Maria Camargo; Martínez-Martínez, Luis; Ocampo-Sosa, Alain Antonio

    2015-01-01

    An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed. PMID:26676375

  1. Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

    PubMed

    Shakibaie, Mojtaba; Forootanfar, Hamid; Golkari, Yaser; Mohammadi-Khorsand, Tayebe; Shakibaie, Mohammad Reza

    2015-01-01

    The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated. PMID:25175509

  2. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    PubMed

    Bi, Dexi; Xie, Yingzhou; Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  3. 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

  4. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene

    PubMed Central

    Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M.; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  5. Synthetic Antimicrobial Peptides Exhibit Two Different Binding Mechanisms to the Lipopolysaccharides Isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae

    PubMed Central

    Chai, Hanbo; Allen, William E.; Hicks, Rickey P.

    2014-01-01

    Circular dichroism and 1H NMR were used to investigate the interactions of a series of synthetic antimicrobial peptides (AMPs) with lipopolysaccharides (LPS) isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae. Previous CD studies with AMPs containing only three Tic-Oic dipeptide units do not exhibit helical characteristics upon interacting with small unilamellar vesicles (SUVs) consisting of LPS. Increasing the number of Tic-Oic dipeptide units to six resulted in five analogues with CD spectra that exhibited helical characteristics on binding to LPS SUVs. Spectroscopic and in vitro inhibitory data suggest that there are two possible helical conformations resulting from two different AMP-LPS binding mechanisms. Mechanism one involves a helical binding conformation where the AMP binds LPS very strongly and is not efficiently transported across the LPS bilayer resulting in the loss of inhibitory activity. Mechanism two involves a helical binding conformation where the AMP binds LPS very loosely and is efficiently transported across the LPS bilayer resulting in an increase in inhibitory activity. Mechanism three involves a nonhelical binding conformation where the AMP binds LPS very loosely and is efficiently transported across the LPS bilayer resulting in an increase in inhibitory activity. PMID:25610647

  6. In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa and Enterobacteriaceae recovered in Spanish medical centres: Results of the CENIT study.

    PubMed

    Tato, Marta; García-Castillo, María; Bofarull, Ana Moreno; Cantón, Rafael

    2015-11-01

    Ceftolozane/tazobactam is a novel antimicrobial agent with activity against Pseudomonas aeruginosa, including drug-resistant strains, and other Gram-negative pathogens, including most extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. The CENIT study evaluated the in vitro activity of ceftolozane/tazobactam and comparators against clinical isolates of P. aeruginosa (n=500) and Enterobacteriaceae (n=500) collected from patients with complicated intra-abdominal, complicated urinary tract, lower respiratory tract or bloodstream infections in 10 medical centres in Spain (January-September 2013). Antimicrobial susceptibility was determined by the ISO broth microdilution method using commercial dry-form panels and results were interpreted per EUCAST and CLSI guidelines and for ceftolozane/tazobactam with FDA criteria. Ceftolozane/tazobactam and ceftolozane alone were the most potent (MIC(50/90), 0.5/4 mg/L) agents tested against all P. aeruginosa isolates. This advantage was maintained regardless of resistance phenotype, even against isolates resistant to multiple antibiotics. Ceftolozane/tazobactam demonstrated excellent overall activity (MIC50/90, 0.25/0.5 mg/L) against all 250 Escherichia coli isolates, including isolates displaying a wild-type (MIC(90), 0.25/0.25 mg/L) or ESBL (MIC(50/90), 0.5/1mg/L) phenotype, and good activity against isolates displaying an AmpC-like phenotype (MIC range 0.25-4 mg/L). Ceftolozane/tazobactam demonstrated good overall activity (MIC(50/90), 0.25/4 mg/L) against all 104 Klebsiella spp. isolates, although activity was lower against those with an ESBL phenotype (MIC(50/90), 4/16 mg/L), and was inactive against the carbapenemase-producing isolates (MIC≥64 mg/L). Ceftolozane/tazobactam demonstrated excellent in vitro activity against most of the P. aeruginosa and Enterobacteriaceae clinical isolates obtained from medical centres in Spain, supporting the potential value of ceftolozane/tazobactam in treating infections due

  7. 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

  8. Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

    PubMed

    Smith, Karen; Rajendran, Ranjith; Kerr, Stephen; Lappin, David F; Mackay, William G; Williams, Craig; Ramage, Gordon

    2015-09-01

    In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis. PMID:26162475

  9. d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

    PubMed Central

    Akers, Kevin S.; Romano, Desiree R.; Woodbury, Ronald L.; Hardy, Sharanda K.; Murray, Clinton K.; Wenke, Joseph C.

    2014-01-01

    Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of d-amino acids (d-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of d-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. d-Met, d-Phe, and d-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined with d-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of d-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of d-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity. PMID:24841260

  10. 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

  11. Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

    PubMed

    Pelin, Marco; Forino, Martino; Brovedani, Valentina; Tartaglione, Luciana; Dell'Aversano, Carmela; Pistocchi, Rossella; Poli, Mark; Sosa, Silvio; Florio, Chiara; Ciminiello, Patrizia; Tubaro, Aurelia

    2016-02-01

    This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program. PMID:26714047

  12. Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells

    PubMed Central

    Stanton, Bruce A.; Coutermarsh, Bonita; Barnaby, Roxanna; Hogan, Deborah

    2015-01-01

    Background P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials. PMID:26018799

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

    PubMed

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

    2016-01-01

    We present the draft genome assembly of an extensively drug-resistant (XDR)Pseudomonas aeruginosastrain 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

  14. [Investigation of the effect of efflux pump inhibitors to MIC values of ciprofloxacin in clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus].

    PubMed

    Cetinkaya, Ebru; Coban, Ahmet Yilmaz; Durupinar, Belma

    2008-10-01

    The aim of this study was to investigate the effects of efflux pump inhibitors on the minimal inhibitory concentration (MIC) values of ciprofloxacin (CIP) in fluoroquinolone-resistant 42 Pseudomonas aeruginosa (n= 42), Escherichia coil (n= 97), Acinetobacter baumannii (n= 58) and Staphylococcus aureus (n= 80) strains isolated from clinical specimens. For this purpose phenylalanyl-arginyl-beta-naphthylamide (PA beta N) was used for P. aeruginosa, E. coli, A. baumannii and reserpine for S. aureus isolates as pump inhibitors. Fluoroquinolone resistance of the clinical isolates were determined by VITEK2 Compact (BioMerieux, France) automated system and confirmed with standard broth microdilution method. For the investigation of the effects of inhibitor agents, the MIC values were also determined in the presence of 25 microg/ml and 100 microg/ml PA beta N and 20 microg/ml reserpine. In the presence of 25 mg/l PA beta N, 61.9% of CIP resistant P. aeruginosa strains converted to susceptible ones, while this rate was 73.8% in the presence of 100 mg/l PA beta N. In A. baumannii clinical isolates, 8.6% and 15.5% of CIP-resistant strains have become susceptible in the presence of 25 mg/l and 100 mg/l PA beta N, respectively. Similarly the MIC values for CIP have decreased > or = 4 folds in 42.2%, and > or = 2 folds in 30.9% of E. coli isolates, in the presence of 25 mg/l PA beta N, however, there was no change in MICs of 26.9% of E. coli strains. The MIC values have also been lowered for > or = 4 folds in 83.6%, and two folds in 13.4% of E. coli strains by the use of 100 mg/l PA beta N concentration, however, no decrease in MIC values was detected in 3% of the isolates. 20 mg/l of reserpine have caused a decrease of > or = 4 folds in 8.75%, and two folds in 33.75% of S. aureus isolates, while there was no change in MIC values of 57.5% of S. aureus strains. Our results showed that PA beta N causes significant reduction in MIC values for CIP in the clinical isolates of P

  15. Pyocyanine Biosynthetic Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa and Detection of Pyocyanine’s Antimicrobial Effects with or without Colloidal Silver Nanoparticles

    PubMed Central

    Nowroozi, Jamileh; Akhavan Sepahi, Abbas; Rashnonejad, Afrooz

    2012-01-01

    Objective: Pyocyanine plays an important role in the pathogenesis of Pseudomonas aeruginosa, (P. aeruginosa) and is known to have inhibitory and bactericidal effects. This study has aimed to detect the phenazine biosynthetic operon (phz ABCDEFG) and two phenazine modifying genes (phzM and phzS) by polymerase chain reaction (PCR) and detection of its possible protein bands by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The antimicrobial effects of pyocyanine alone and mixed with colloidal silver nanoparticles were studied. Materials and Methods: In this descriptive study, clinical and environmental species of P. aeruginosa were isolated by thioglycollate medium culture and cetrimide agar, respectively. The existence of a phenazine biosynthetic operon and two phenazine modifying genes as well as their protein products were confirmed by PCR and SDS-PAGE, respectively. Pyocyanine was extracted with chloroform and its antimicrobial effects against bacteria such as; Escherichia coli (E. coli), P. aeruginosaand Staphylococcus aureus (S. aureus) bacteria and yeast Candida albicans (C. albicans) were tested using well, spot and disk diffusion methods. Results: In this study, 3 out of 48 clinical strains were unable to produce pyocyanine on cetrimide and Mueller Hinton (MH) agar. Two strains did not have phenazine modifying gene bands. Another strain did not have the possible protein band of the phzM gene. Pyocyanine had antimicrobial effects against the microbial strains, which increased in the presence of silver nanoparticles. Conclusion: According to the results of the present study, some P. aeruginosa strains are unable to produce pyocyanine due to the absence of the phzM or phzS genes. Therefore, these genes have an important role in pyocyanine production in P. aeruginosa. Pyocyanine shows synergistic antimicrobial effects in the presence of silver nanoparticles against microbial strains. PMID:23626932

  16. Identification of plasmid OXA and other β-lactamase genes among carbapenem-resistant isolates of Pseudomonas aeruginosa from the Clinical University Hospital in northeastern Poland.

    PubMed

    Sacha, Paweł; Michalska, Anna; Ojdana, Dominika; Wieczorek, Piotr; Hauschild, Tomasz; Majewski, Piotr; Tryniszewska, Elżbieta

    2015-04-01

    The aim of the study was to evaluate the prevalence of OXA and other β-lactamase genes, antibiotic susceptibility, and the genetic relatedness among clinical isolates of P. aeruginosa resistant to carbapenems. The presence of bla- OXA genes was demonstrated in 48% of isolates belonging to four PFGE profiles. Most of them contained the blaOXA-2 gene (88.3%). Other blaOXA genes (Ps1310 with blaOXA-30 and Ps1309 with blaOXA-10) were found in only two isolates. The tested isolates also contained other β-lactamase genes such as blaVIM-2, blaVIM-4, blaSHV-5, and blaTEM-1. All isolates were susceptible only to colistin (100%). PMID:25938753

  17. Unexpected Challenges in Treating Multidrug-Resistant Gram-Negative Bacteria: Resistance to Ceftazidime-Avibactam in Archived Isolates of Pseudomonas aeruginosa

    PubMed Central

    Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Hujer, Andrea M.; Domitrovic, T. Nicholas; Hujer, Kristine M.; Hurless, Kelly N.; Tuohy, Marion; Hall, Geraldine

    2014-01-01

    Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors. PMID:25451057

  18. Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water

    PubMed Central

    Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

    2015-01-01

    The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

  19. Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

    PubMed

    Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

    2015-01-01

    The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

  20. Isolation of a non-fermentative bacterium, Pseudomonas aeruginosa, using intracellular carbon for denitrification and phosphorus-accumulation and relevant metabolic mechanisms.

    PubMed

    Liu, Hui; Wang, Qin; Sun, Yanfu; Zhou, Kangqun; Liu, Wen; Lu, Qian; Ming, Caibing; Feng, Xidan; Du, Jianjun; Jia, Xiaoshan; Li, Jun

    2016-07-01

    A newly designed pilot-scale system was developed to enrich denitrifying phosphate-accumulating organisms (DNPAOs) for nitrogen and phosphorus nutrient removal synchronously. A strain of DNPAOs was isolated and its biochemical characteristics and metabolic mechanisms of this bacterial strain were analyzed. The results showed that compared with previously reported system, this newly designed system has higher removal rates of nutrients. Removal efficiencies of NH3-N, TN, TP, and COD in actual wastewater were 82.64%, 79.62%, 87.22%, and 90.41%, respectively. Metabolic activity of DNPAOs after anoxic stage in this study even reached 94.64%. Pseudomonas aeruginosa is a strain of non-fermentative DNPAOs with strong nitrogen and phosphorus removal abilities. Study on the metabolic mechanisms suggested that intracellular PHB of P. aeruginosa plays dual roles, supplying energy for phosphorus accumulation and serving as a major carbon source for denitrification. PMID:26995616

  1. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France.

    PubMed

    Poirel, L; Naas, T; Nicolas, D; Collet, L; Bellais, S; Cavallo, J D; Nordmann, P

    2000-04-01

    Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to beta-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing beta-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B beta-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, including P. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 beta-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 beta-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-beta-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 microM). VIM-2 conferred a resistance pattern to beta-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime. bla(VIM-2) was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. bla(VIM-2) was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the bla(VIM-1)-containing

  2. Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions.

    PubMed

    Yoon, Sang Sun; Coakley, Ray; Lau, Gee W; Lymar, Sergei V; Gaston, Benjamin; Karabulut, Ahmet C; Hennigan, Robert F; Hwang, Sung-Hei; Buettner, Garry; Schurr, Michael J; Mortensen, Joel E; Burns, Jane L; Speert, David; Boucher, Richard C; Hassett, Daniel J

    2006-02-01

    Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways. PMID:16440061

  3. Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions

    PubMed Central

    Yoon, Sang Sun; Coakley, Ray; Lau, Gee W.; Lymar, Sergei V.; Gaston, Benjamin; Karabulut, Ahmet C.; Hennigan, Robert F.; Hwang, Sung-Hei; Buettner, Garry; Schurr, Michael J.; Mortensen, Joel E.; Burns, Jane L.; Speert, David; Boucher, Richard C.; Hassett, Daniel J.

    2006-01-01

    Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2–) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2– also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways. PMID:16440061

  4. 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

  5. Effects of linear alkylbenzene sulfonate on the growth and toxin production of Microcystis aeruginosa isolated from Lake Dianchi.

    PubMed

    Wang, Zhi; Zhang, Junqian; Song, Lirong; Li, Enhua; Wang, Xuelei; Xiao, Bangding

    2015-04-01

    The exogenous organic pollutant linear alkylbenzene sulfonate (LAS) pollution and Microcystis bloom are two common phenomena in eutrophic lakes, but the effects of LAS alone on Microcystis remain unclear. In the present study, we investigated the effects of LAS on the growth, photochemical efficiency, and microcystin production of Microcystis aeruginosa in the laboratory. Results showed that low LAS (≤10 mg/L) concentrations improved the growth of M. aeruginosa (12 days of exposure). High LAS (20 mg/L) concentrations inhibited the growth of M. aeruginosa on the first 8 days of exposure; afterward, growth progressed. After 12 days of exposure, the concentrations of chlorophyll a in algal cells were not significantly affected by any of LAS concentrations (0.05 to 20 mg/L) in the present study; by contrast, carotenoid and protein concentrations were significantly inhibited when LAS concentrations reached as high as 20 mg/L. After 6 and 12 days of exposure, low LAS (≤10 mg/L) concentrations enhanced the maximal photochemical efficiency (Fv/Fm) and the maximal electron transport rate (ETRmax) of M. aeruginosa. Furthermore, LAS increased the microcystin production of M. aeruginosa. Extracellular and intracellular microcystin contents were significantly increased after M. aeruginosa was exposed to high LAS concentrations. Our results indicated that LAS in eutrophic lakes may increase the risk of Microcystis bloom and microcystin production. PMID:25382498

  6. Additional Insights on the Bastadins – Isolation of Analogs from the Sponge Ianthella cf. reticulata and Exploration of the Oxime Configurations†

    PubMed Central

    Calcul, Laurent; Inman, Wayne D.; Morris, Alexi A.; Tenney, Karen; Ratnam, Joseline; McKerrow, James H.; Valeriote, Frederick A.; Crews, Phillip

    2015-01-01

    The focus of this study was on the bastadin class of bromotyrosine derivatives, commonly isolated from Ianthella marine sponges, and is the first report on the secondary metabolites from Ianthella cf. reticulata. Two new bastadins were isolated, (E,Z)-bastadin 19 (1b), a diastereoisomer of the known (E,E)-bastadin 19 (1a), and dioxepine bastadin 3 (2), an unusual dibenzo-1,3-dioxepine. A bastadin NMR database was created and assisted in the structure determination of 1b, 2 and the rapid dereplication of ten other known compounds including bastadins 2–9 (3-10), 13 (11) and 19 (1a). The geometry of the 2-(hydroxyimino)-N-alkylamide chains, a chemical feature present in all bastadins, was further probed and new insights regarding the natural oxime configuration are discussed. Bastadins possessing (E,Z), (Z,E) or (E,E)-dioxime configurations could be artifacts of isolation or storage in solution. Therefore, this point was explored by photochemical and thermal isomerization studies, as well as molecular mechanics calculations. Bastadins 13 (11) and 19 (1a) exhibited moderate inhibition against Trypanosoma brucei and bastadin 4 (5) was cytotoxic to HCT-116 colon cancer cells. PMID:20102170

  7. Antimicrobial Activity of Fosfomycin-Tobramycin Combination against Pseudomonas aeruginosa Isolates Assessed by Time-Kill Assays and Mutant Prevention Concentrations

    PubMed Central

    Díez-Aguilar, María; Tedim, Ana P.; Rodríguez, Irene; Aktaş, Zerrin; Cantón, Rafael

    2015-01-01

    The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 μg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 μg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 μg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 μg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 μg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 μg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 μg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments. PMID:26195514

  8. Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium

    PubMed Central

    Ferreira, Jose A. G.; Penner, John C.; Moss, Richard B.; Haagensen, Janus A. J.; Clemons, Karl V.; Spormann, Alfred M.; Nazik, Hasan; Cohen, Kevin; Banaei, Niaz; Carolino, Elisabete; Stevens, David A.

    2015-01-01

    Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment. PMID:26252384

  9. Correlation between cytotoxicity induced by Pseudomonas aeruginosa clinical isolates from acute infections and IL-1β secretion in a model of human THP-1 monocytes.

    PubMed

    Anantharajah, Ahalieyah; Buyck, Julien M; Faure, Emmanuel; Glupczynski, Youri; Rodriguez-Villalobos, Hector; De Vos, Daniel; Pirnay, Jean-Paul; Bilocq, Florence; Guery, Benoît; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Bambeke, Françoise

    2015-10-01

    Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express ExoU. PMID:26203053

  10. A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase

    PubMed Central

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Shiroma, Akino; Nakano, Kazuma; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi; Shimojima, Masahiro; Kirikae, Teruo

    2016-01-01

    A carbapenem-resistant strain of Pseudomonas aeruginosa, NCGM1984, was isolated in 2012 from a hospitalized patient in Japan. Immunochromatographic assay showed that the isolate was positive for IMP-type metallo-β-lactamase. Complete genome sequencing revealed that NCGM1984 harbored two copies of blaIMP-34, located at different sites on the chromosome. Each blaIMP-34 was present in the same structures of the class 1 integrons, tnpA(ISPa7)-intI1-qacG-blaIMP-34-aac(6')-Ib-qacEdelta1-sul1-orf5-tniBdelta-tniA. The isolate belonged to multilocus sequence typing ST235, one of the international high-risk clones. IMP-34, with an amino acid substitution (Glu126Gly) compared with IMP-1, hydrolyzed all β-lactamases tested except aztreonam, and its catalytic activities were similar to IMP-1. This is the first report of a clinical isolate of an IMP-34-producing P. aeruginosa harboring two copies of blaIMP-34 on its chromosome. PMID:27055243

  11. Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs.

    PubMed

    Cramer, Nina; Klockgether, Jens; Wrasman, Kristie; Schmidt, Mario; Davenport, Colin F; Tümmler, Burkhard

    2011-07-01

    Clones C and PA14 are the worldwide most abundant clonal complexes in the Pseudomonas aeruginosa population. The microevolution of clones C and PA14 was investigated in serial cystic fibrosis (CF) airway isolates collected over 20 years since the onset of colonization. Intraclonal evolution in CF lungs was resolved by genome sequencing of first, intermediate and late isolates and subsequent multimarker SNP genotyping of the whole strain panel. Mapping of sequence reads onto the P. aeruginosa PA14 reference genome unravelled an intraclonal and interclonal sequence diversity of 0.0035% and 0.68% respectively. Clone PA14 diversified into three branches in the patient's lungs, and the PA14 population acquired 15 nucleotide substitutions and a large deletion during the observation period. The clone C genome remained invariant during the first 3 years in CF lungs; however, 15 years later 947 transitions and 12 transversions were detected in a clone C mutL mutant strain. Key mutations occurred in retS, RNA polymerase, multidrug transporter, virulence and denitrification genes. Late clone C and PA14 persistors in the CF lungs were compromised in growth and cytotoxicity, but their mutation frequency was normal even in mutL mutant clades. PMID:21492363

  12. Inhibition of High-Mobility Group Box 1 Protein (HMGB1) Enhances Bacterial Clearance and Protects against Pseudomonas Aeruginosa Pneumonia in Cystic Fibrosis

    PubMed Central

    Entezari, Maria; Weiss, Daniel J; Sitapara, Ravikumar; Whittaker, Laurie; Wargo, Matthew J; Li, JianHua; Wang, Haichao; Yang, Huan; Sharma, Lokesh; Phan, Binh D; Javdan, Mohammad; Chavan, Sangeeta S; Miller, Edmund J; Tracey, Kevin J; Mantell, Lin L

    2012-01-01

    Pulmonary infection with Pseudomonas aeruginosa and neutrophilic lung inflammation significantly contribute to morbidity and mortality in cystic fibrosis (CF). High-mobility group box 1 protein (HMGB1), a ubiquitous DNA binding protein that promotes inflammatory tissue injury, is significantly elevated in CF sputum. However, its mechanistic and potential therapeutic implications in CF were previously unknown. We found that HMGB1 levels were significantly elevated in bronchoalveolar lavage fluids (BALs) of CF patients and cystic fibrosis transmembrane conductance regulator (CFTR )−/− mice. Neutralizing anti-HMGB1 monoclonal antibody (mAb) conferred significant protection against P. aeruginosa–induced neutrophil recruitment, lung injury and bacterial infection in both CFTR−/− and wild-type mice. Alveolar macrophages isolated from mice treated with anti-HMGB1 mAb had improved phagocytic activity, which was suppressed by direct exposure to HMGB1. In addition, BAL from CF patients significantly impaired macrophage phagocytotic function, and this impairment was attenuated by HMGB1-neutralizing antibodies. The HMGB1-mediated suppression of bacterial phagocytosis was attenuated in macrophages lacking toll-like receptor (TLR)-4, suggesting a critical role for TLR4 in signaling HMGB1-mediated macrophage dysfunction. These studies demonstrate that the elevated levels of HMGB1 in CF airways are critical for neutrophil recruitment and persistent presence of P. aeruginosa in the lung. Thus, HMGB1 may provide a therapeutic target for reducing bacterial infection and lung inflammation in CF. PMID:22314397

  13. Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

    PubMed

    Hoshino, T; Kose-Terai, K; Uratani, Y

    1991-03-01

    The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II. PMID:1900503

  14. Antibacterial activity of liposomal gentamicin against Pseudomonas aeruginosa: a time-kill study.

    PubMed

    Rukholm, Gavin; Mugabe, Clement; Azghani, Ali O; Omri, Abdelwahab

    2006-03-01

    Cystic fibrosis (CF) is a common and lethal genetic disorder with a carrier frequency of 1 in 29 Caucasians. Chronic respiratory infections with Pseudomonas aeruginosa are the leading cause of morbidity and mortality in individuals with CF. Aminoglycoside antibiotics, including gentamicin, are highly effective against P. aeruginosa, but severe toxicity limits their use. One potential strategy for avoiding this problem is to encapsulate aminoglycosides in liposomes. In this study, we compared the bactericidal capacity of liposome-encapsulated gentamicin with that of free antibiotic against clinical isolates of P. aeruginosa. Liposome size, encapsulation efficiency and minimal inhibitory concentrations (MICs) of the free and liposomal gentamicin against gentamicin-sensitive and -resistant strains of P. aeruginosa were determined. In vitro time-kill studies were performed using free and liposomal gentamicin at 1, 2 or 4 times the MICs. The average liposomal size was 426.25 +/- 13.56 nm, with a gentamicin encapsulation efficiency of 4.51 +/- 0.54%. The MICs for liposomal gentamicin were significantly lower than those of corresponding free gentamicin. In addition, the time-kill values for liposomal gentamicin were either equivalent to or better than those of the free antibiotic. In conclusion, our liposomal gentamicin formulation is a more potent antipseudomonal drug with an improved killing time and prolonged antimicrobial activity. PMID:16472992

  15. 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

  16. NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress.

    PubMed

    Ketelboeter, Laura M; M Ketelboeter, Laura; Potharla, Vishwakanth Y; Y Potharla, Vishwakanth; Bardy, Sonia L; L Bardy, Sonia

    2014-09-01

    Pyomelanin is a brown/black extracellular pigment with antioxidant and iron acquisition properties that is produced by a number of different bacteria. Production of pyomelanin in Pseudomonas aeruginosa contributes to increased resistance to oxidative stress and persistence in chronic infections. We demonstrate that pyomelanin production can be inhibited by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC). This treatment increases sensitivity of pyomelanogenic P. aeruginosa strains to oxidative stress, without altering the growth rate or resistance to aminoglycosides. As such, NTBC has potential to function as an anti-virulence factor in treating pyomelanogenic bacterial infections. PMID:24801336

  17. CF Mutation Panel

    MedlinePlus

    ... page: Was this page helpful? Also known as: Cystic Fibrosis Genotyping; CF DNA Analysis; CF Gene Mutation Panel; CF Molecular Genetic Testing Formal name: Cystic Fibrosis Gene Mutation Panel Related tests: Sweat Test ; Trypsinogen ; ...

  18. 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

  19. 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

  20. 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

  1. 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

  2. Molecular epidemiology of clinical Pseudomonas aeruginosa isolates carrying IMP-1 metallo-beta-lactamase gene in a University Hospital in Turkey.

    PubMed

    Ozgumus, Osman Birol; Caylan, Rahmet; Tosun, Ilknur; Sandalli, Cemal; Aydin, Kemalettin; Koksal, Iftihar

    2007-01-01

    Pseudomonas aeruginosa isolates carrying IMP- or VIM-type metallo-beta-lactamase (MBL) have been increasingly reported in hospitals worldwide. One hundred P. aeruginosa clinical isolates from unrelated inpatients hospitalized at a Turkish university hospital were screened for the presence of bla(IMP) and bla(VIM) genes by polymerase chain reaction (PCR). One (1%) isolate was found to carry a VIM-type MBL gene, whereas nine (9%) carried an IMP-1 MBL gene carried on a cassette inserted into a class 1 integron. Only four of the IMP producers were detected as MBL producers according to E-test MBL. Minimum inhibitory concentrations (MICs) of imipenem for the IMP-1 and VIM-type MBL-producers were highly variable (MIC values, 8-128 mug/ml). Imipenem resistance was not plasmid-mediated according to the transformation assays. Piperacillin/tazobactam was the only effective drug in antimicrobial susceptibility testing. No aztreonam-resistant IMP and VIM producers were detected to produce an extended-spectrum beta-lactamase (ESBL). Three class 1 integrons of approximately 2,300 bp, 1,800 bp, and 1,500 bp in size were detected in each of the nine IMP-positive isolates. Sequencing revealed three novel gene cassette arrays, aac(3)-1c-cmlA5, bla(IMP-1)-aadA7-like, and aacA7-smr-2-orfD. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) indicated that a clonal spread of IMP-1-producers had occurred in this hospital. PMID:17949306

  3. Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model.

    PubMed Central

    Howe, T R; Iglewski, B H

    1984-01-01

    Mutants of Pseudomonas aeruginosa are described which are markedly deficient in alkaline protease production. Characterization of these mutants in vitro suggests that the mutations in two of these strains are specific for alkaline protease production. Examination of these mutants in a mouse eye model demonstrates that alkaline protease is required for the establishment of corneal infections with P. aeruginosa PA103. Mutants deficient in alkaline protease production could not colonize traumatized cornea and did not produce the corneal damage characteristic of infection by the parental strain. Addition of subdamaging amounts of alkaline protease to eyes infected with the protease-deficient mutants resulted in infections which were indistinguishable from infections caused by the parental strain. PMID:6421735

  4. Novel 6′-N-Aminoglycoside Acetyltransferase AAC(6′)-Iaj from a Clinical Isolate of Pseudomonas aeruginosa

    PubMed Central

    Tada, Tatsuya; Miyoshi-Akiyama, Tohru; Shimada, Kayo; Shimojima, Masahiro

    2013-01-01

    Pseudomonas aeruginosa NCGM1588 has a novel chromosomal class 1 integron, In151, which includes the aac(6′)-Iaj gene. The encoded protein, AAC(6′)-Iaj, was found to consist of 184 amino acids, with 70% identity to AAC(6′)-Ia. Escherichia coli transformed with a plasmid containing the aac(6′)-Iaj gene acquired resistance to all aminoglycosides tested except gentamicin. Of note, aac(6′)-Iaj contributed to the resistance to arbekacin. Thin-layer chromatography revealed that AAC(6′)-Iaj acetylated all aminoglycosides tested except gentamicin. These findings indicated that AAC(6′)-Iaj is a functional acetyltransferase that modifies the amino groups at the 6′ positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin. PMID:23070167

  5. Low-level resistance and clonal diversity of Pseudomonas aeruginosa among chronically colonized cystic fibrosis patients.

    PubMed

    Ferreira, Alex Guerra; Leão, Robson Souza; Carvalho-Assef, Ana Paula D'alincourt; da Silva, Érica Aparecida dos Santos Ribeiro; Firmida, Monica de Cássia; Folescu, Tania Wrobel; Paixão, Vilma Almeida; Santana, Maria Angélica; de Abreu e Silva, Fernando Antonio; Barth, Afonso Luís; Marques, Elizabeth Andrade

    2015-12-01

    A prospective study was conducted in Brazil to evaluate antimicrobial resistance patterns and molecular epidemiology of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients with chronic lung infection. All isolates were obtained between May 2009 and June 2010 from 75 patients seen in four reference centers in Brazil: HCPA (20 patients) and HEOM (15 patients), located in southern and northeastern Brazil, respectively; IFF (20 patients) and HUPE (20 patients), both in southwestern Brazil. Antimicrobial susceptibility testing, PCR for detection of carpapenemases, and pulsed-field gel electrophoresis (PFGE) were performed in 274 isolates. A total of 224 PFGE types were identified and no clones were found circulating among the centers or within the same center. Despite the chronic infection, most patients were colonized by intermittent clones. Only three patients (4%) maintained the same clone during the study. The resistance rates were lower than 30% for the majority of antimicrobials tested in all centers and only 17% of isolates were multiresistant. Isolates (n = 54) with reduced susceptibility to imipenem and/or meropenem presented negative results for blaSPM-1, blaIMP-1, blaVIM , and blaKPC genes. Our results indicate an unexpected low level of antimicrobial resistance and a high genotypic diversity among P. aeruginosa from Brazilian chronic CF patients. PMID:26522829

  6. Coexistence and within-host evolution of diversified lineages of hypermutable Pseudomonas aeruginosa in long-term cystic fibrosis infections.

    PubMed

    Feliziani, Sofía; Marvig, Rasmus L; Luján, Adela M; Moyano, Alejandro J; Di Rienzo, Julio A; Krogh Johansen, Helle; Molin, Søren; Smania, Andrea M

    2014-10-01

    The advent of high-throughput sequencing techniques has made it possible to follow the genomic evolution of pathogenic bacteria by comparing longitudinally collected bacteria sampled from human hosts. Such studies in the context of chronic airway infections by Pseudomonas aeruginosa in cystic fibrosis (CF) patients have indicated high bacterial population diversity. Such diversity may be driven by hypermutability resulting from DNA mismatch repair system (MRS) deficiency, a common trait evolved by P. aeruginosa strains in CF infections. No studies to date have utilized whole-genome sequencing to investigate within-host population diversity or long-term evolution of mutators in CF airways. We sequenced the genomes of 13 and 14 isolates of P. aeruginosa mutator populations from an Argentinian and a Danish CF patient, respectively. Our collection of isolates spanned 6 and 20 years of patient infection history, respectively. We sequenced 11 isolates from a single sample from each patient to allow in-depth analysis of population diversity. Each patient was infected by clonal populations of bacteria that were dominated by mutators. The in vivo mutation rate of the populations was ∼100 SNPs/year-∼40-fold higher than rates in normo-mutable populations. Comparison of the genomes of 11 isolates from the same sample showed extensive within-patient genomic diversification; the populations were composed of different sub-lineages that had coexisted for many years since the initial colonization of the patient. Analysis of the mutations identified genes that underwent convergent evolution across lineages and sub-lineages, suggesting that the genes were targeted by mutation to optimize pathogenic fitness. Parallel evolution was observed in reduction of overall catabolic capacity of the populations. These findings are useful for understanding the evolution of pathogen populations and identifying new targets for control of chronic infections. PMID:25330091

  7. Coexistence and Within-Host Evolution of Diversified Lineages of Hypermutable Pseudomonas aeruginosa in Long-term Cystic Fibrosis Infections

    PubMed Central

    Feliziani, Sofía; Moyano, Alejandro J.; Di Rienzo, Julio A.; Krogh Johansen, Helle; Molin, Søren; Smania, Andrea M.

    2014-01-01

    The advent of high-throughput sequencing techniques has made it possible to follow the genomic evolution of pathogenic bacteria by comparing longitudinally collected bacteria sampled from human hosts. Such studies in the context of chronic airway infections by Pseudomonas aeruginosa in cystic fibrosis (CF) patients have indicated high bacterial population diversity. Such diversity may be driven by hypermutability resulting from DNA mismatch repair system (MRS) deficiency, a common trait evolved by P. aeruginosa strains in CF infections. No studies to date have utilized whole-genome sequencing to investigate within-host population diversity or long-term evolution of mutators in CF airways. We sequenced the genomes of 13 and 14 isolates of P. aeruginosa mutator populations from an Argentinian and a Danish CF patient, respectively. Our collection of isolates spanned 6 and 20 years of patient infection history, respectively. We sequenced 11 isolates from a single sample from each patient to allow in-depth analysis of population diversity. Each patient was infected by clonal populations of bacteria that were dominated by mutators. The in vivo mutation rate of the populations was ∼100 SNPs/year–∼40-fold higher than rates in normo-mutable populations. Comparison of the genomes of 11 isolates from the same sample showed extensive within-patient genomic diversification; the populations were composed of different sub-lineages that had coexisted for many years since the initial colonization of the patient. Analysis of the mutations identified genes that underwent convergent evolution across lineages and sub-lineages, suggesting that the genes were targeted by mutation to optimize pathogenic fitness. Parallel evolution was observed in reduction of overall catabolic capacity of the populations. These findings are useful for understanding the evolution of pathogen populations and identifying new targets for control of chronic infections. PMID:25330091

  8. 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

  9. Whole, submandibular, and parotid saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis.

    PubMed Central

    Komiyama, K; Habbick, B F; Tumber, S K

    1989-01-01

    The aggregation of mucoid and nonmucoid Pseudomonas aeruginosa by submandibular, parotid, and whole saliva from patients with cystic fibrosis (CF) and non-CF subjects was investigated. There were significant differences (P less than 0.01) in aggregation of mucoid and nonmucoid variants of P. aeruginosa by submandibular and whole saliva from CF patients and non-CF subjects. However, the differences in the parotid secretion were not as pronounced. Patients with CF who were colonized with P. aeruginosa demonstrated a significantly higher (P less than 0.05) percent aggregation of the mucoid variants by the submandibular secretion and of both mucoid and nonmucoid variants by whole saliva, compared with corresponding secretions from patients with CF not colonized with this pathogen. The parotid saliva aggregation activity was not markedly different for the two groups with CF. From patients with CF, whole saliva demonstrated a higher percent P. aeruginosa aggregation than did the submandibular saliva. In non-CF subjects, however, the percent aggregation of P. aeruginosa by submandibular saliva was higher than that by whole saliva. Our results indicate that the sero-mucous products of the submandibular gland have a more significant role in P. aeruginosa aggregation than the serous secreting parotid cells and that the submandibular secretion is possibly responsible for the differences in oral colonization by this pathogen in subjects with and without CF. PMID:2494114

  10. Predominance of carbapenem-resistant Pseudomonas aeruginosa isolates carrying blaIMP and blaVIM metallo-β-lactamases in a major hospital in Costa Rica.

    PubMed

    Toval, Francisco; Guzmán-Marte, Anel; Madriz, Vivian; Somogyi, Teresita; Rodríguez, César; García, Fernando

    2015-01-01

    This study aimed to assess the molecular basis of the resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa recovered from a tertiary-level health facility in San José, Costa Rica. A total of 198 non-duplicated isolates were evaluated for their susceptibility to β-lactams, aminoglycosides and fluoroquinolones. The production of metallo-β-lactamases (MBLs), the presence of MBL encoding genes (blaIMP, blaVIM and blaGIM-1) and the occurrence of these genes within class 1 integrons were investigated. In addition, an ERIC2 PCR fingerprinting method was used to elucidate the distribution of the detected MBL genes within the strain collection. Of the 198 isolates tested, 125 (63.1 %) were categorized as carbapenem-resistant. The majority (88.8 %) of the carbapemen-resistant isolates also showed resistance to ceftazidime, cefepime, aztreonam, ticarcillin/clavulanic acid, amikacin, gentamicin, tobramycin, ciprofloxacin and gatifloxacin. Among the carbapenem-resistant isolates, 102 (81.6 %) showed MBL activity. Strikingly, both blaIMP and blaVIM genes were simultaneously detected in most (94.1 %) of the 102 MBL producers. Five carbapenem-resistant MBL producers were positive only for blaIMP genes. Almost 70 % of the isolates examined harboured the intI1 gene, accompanied by the sul1 and qacEΔ1 genes in 136 (99 %) and 122 (89 %) isolates, respectively. The majority (94.4 %) of the carbapenem-resistant isolates carried the intI1 gene, in contrast to 26 % of the carbapenem-susceptible isolates. Ninety-three out of 96 (96.9 %) isolates carrying both blaIMP and blaVIM genes also harboured the intI1, sul1 and qacEΔ1 genes. Gene cassettes from carbapenem-susceptible and MBL-negative carbapenem-resistant isolates encoded aminoglycoside-resistance enzymes (aadA2, aadA4 and aadA6) as well as orfD and qacF genes. RAPD analysis distributed 126 of the isolates in 29 clusters. Eighty of the 90 blaIMP (+) blaVIM (+) isolates were sorted into 16

  11. In vitro potentiation of carbapenems with ME1071, a novel metallo-beta-lactamase inhibitor, against metallo-beta-lactamase- producing Pseudomonas aeruginosa clinical isolates.

    PubMed

    Ishii, Yoshikazu; Eto, Maki; Mano, Yoko; Tateda, Kazuhiro; Yamaguchi, Keizo

    2010-09-01

    ME1071, a maleic acid derivative, is a novel specific inhibitor for metallo-beta-lactamases (MBL). In this study, the potentiation of ME1071 in combination with several beta-lactams was evaluated using MBL-producing Pseudomonas aeruginosa isolates. The rates of susceptibility of MBL producers to carbapenems (imipenem, biapenem, and doripenem) and ceftazidime were increased by 8 to 27% in the presence of 32 microg/ml of ME1071. The corresponding resistance rates were decreased by 13 to 46%, respectively. On the other hand, ME1071 showed weaker or no potentiation with non-MBL producers. The K(i) value of ME1071 for IMP-1 was 0.4 microM, significantly lower than the K(m) values of carbapenems for the IMP-1 enzyme. On the other hand, the K(i) value of ME1071 for VIM-2 was 120 microM, higher than the K(m) values of carbapenems for the VIM-2 enzyme. Results of this study indicate that ME1071 can potentiate the activity of ceftazidime and carbapenems against MBL-producing strains of P. aeruginosa. PMID:20606062

  12. Within-Host Evolution of the Dutch High-Prevalent Pseudomonas aeruginosa Clone ST406 during Chronic Colonization of a Patient with Cystic Fibrosis.

    PubMed

    van Mansfeld, Rosa; de Been, Mark; Paganelli, Fernanda; Yang, Lei; Bonten, Marc; Willems, Rob

    2016-01-01

    This study investigates adaptation of ST406, a prevalent P. aeruginosa clone, present in 15% of chronically infected cystic fibrosis (CF) patients in the Netherlands, in a newly infected CF patient during three years using whole genome sequencing (WGS), transcriptomics, and phenotypic assays, including biofilm formation. WGS-based phylogeny demonstrates that ST406 is genetically distinct from other reported CF related strains or epidemic clones. Comparative genomic analysis of the early (S1) and late (S2) isolate yielded 42 single nucleotide polymorphisms (SNPs) and 10 indels and a single 7 kb genomic fragment only found in S2. Most SNPs and differentially expressed genes encoded proteins involved in metabolism, secretion and signal transduction or transcription. SNPs were identified in regulator genes mexT and exsA and coincided with differential gene expression of mexE and mexF, encoding the MexE/F efflux pump, genes encoding the type six secretion system (T6SS) and type three secretion system (T3SS), which have also been previously implicated in adaptation of other P. aeruginosa strains during chronic infection of CF lungs. The observation that genetically different strains from different patients have accumulated similar genetic adaptations supports the concept of adaptive parallel evolution of P. aeruginosa in chronically infected CF patients. Phenotypically, there was loss of biofilm maturation coinciding with a significant lower level of transcription of both bfmR and bfmS during chronic colonization. These data suggest that the high-prevalent Dutch CF clone ST406 displays adaptation to the CF lung niche, which involves a limited number of mutations affecting regulators controlling biofilm formation and secretion and genes involved in metabolism. These genes could provide good targets for anti-pseudomonal therapy. PMID:27337151

  13. Within-Host Evolution of the Dutch High-Prevalent Pseudomonas aeruginosa Clone ST406 during Chronic Colonization of a Patient with Cystic Fibrosis

    PubMed Central

    van Mansfeld, Rosa; de Been, Mark; Paganelli, Fernanda; Yang, Lei; Bonten, Marc; Willems, Rob

    2016-01-01

    This study investigates adaptation of ST406, a prevalent P. aeruginosa clone, present in 15% of chronically infected cystic fibrosis (CF) patients in the Netherlands, in a newly infected CF patient during three years using whole genome sequencing (WGS), transcriptomics, and phenotypic assays, including biofilm formation. WGS-based phylogeny demonstrates that ST406 is genetically distinct from other reported CF related strains or epidemic clones. Comparative genomic analysis of the early (S1) and late (S2) isolate yielded 42 single nucleotide polymorphisms (SNPs) and 10 indels and a single 7 kb genomic fragment only found in S2. Most SNPs and differentially expressed genes encoded proteins involved in metabolism, secretion and signal transduction or transcription. SNPs were identified in regulator genes mexT and exsA and coincided with differential gene expression of mexE and mexF, encoding the MexE/F efflux pump, genes encoding the type six secretion system (T6SS) and type three secretion system (T3SS), which have also been previously implicated in adaptation of other P. aeruginosa strains during chronic infection of CF lungs. The observation that genetically different strains from different patients have accumulated similar genetic adaptations supports the concept of adaptive parallel evolution of P. aeruginosa in chronically infected CF patients. Phenotypically, there was loss of biofilm maturation coinciding with a significant lower level of transcription of both bfmR and bfmS during chronic colonization. These data suggest that the high-prevalent Dutch CF clone ST406 displays adaptation to the CF lung niche, which involves a limited number of mutations affecting regulators controlling biofilm formation and secretion and genes involved in metabolism. These genes could provide good targets for anti-pseudomonal therapy. PMID:27337151

  14. Vaccination against respiratory Pseudomonas aeruginosa infection

    PubMed Central

    Grimwood, Keith; Kyd, Jennelle M; Owen, Suzzanne J; Massa, Helen M; Cripps, Allan W

    2014-01-01

    Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection. PMID:25483510

  15. Azithromycin May Antagonize Inhaled Tobramycin When Targeting Pseudomonas aeruginosa in Cystic Fibrosis

    PubMed Central

    Nick, Jerry A.; Moskowitz, Samuel M.; Chmiel, James F.; Forssén, Anna V.; Kim, Sun Ho; Saavedra, Milene T.; Saiman, Lisa; Taylor-Cousar, Jennifer L.

    2014-01-01

    Rationale: Recent studies of inhaled tobramycin in subjects with cystic fibrosis (CF) find less clinical improvement than previously observed. Nonhuman data suggest that in some strains of Pseudomonas aeruginosa, azithromycin can antagonize tobramycin. Objectives: We tested the hypothesis that concomitant azithromycin use correlates with less improvement in key outcome measures in subjects receiving inhaled tobramycin while not affecting those receiving a comparative, nonaminoglycoside inhaled antibiotic. Methods: We studied a cohort of 263 subjects with CF enrolled in a recent clinical trial comparing inhaled tobramycin with aztreonam lysine. We performed a secondary analysis to examine key clinical and microbiologic outcomes based on concomitant, chronic azithromycin use at enrollment. Measurements and Main Results: The cohort randomized to inhaled tobramycin and reporting azithromycin use showed a significant decrease in the percent predicted FEV1 after one and three courses of inhaled tobramycin when compared with those not reporting azithromycin use (28 d: −0.51 vs. 3.43%, P < 0.01; 140 d: −1.87 vs. 6.07%, P < 0.01). Combined azithromycin and inhaled tobramycin use was also associated with earlier need for additional antibiotics, lesser improvement in disease-related quality of life, and a trend toward less reduction in sputum P. aeruginosa density. Subjects randomized to inhaled aztreonam lysine had significantly greater improvement in these outcome measures, which were unaffected by concomitant azithromycin use. Outcomes in those not using azithromycin who received inhaled tobramycin were not significantly different from subjects receiving aztreonam lysine. Azithromycin also antagonized tobramycin but not aztreonam lysine in 40% of P. aeruginosa clinical isolates tested in vitro. Conclusions: Oral azithromycin may antagonize the therapeutic benefits of inhaled tobramycin in subjects with CF with P. aeruginosa airway infection. PMID:24476418

  16. Trifluoromethoxycarbonyl peroxynitrate, CF3OCOOONO2.

    PubMed

    von Ahsen, Stefan; García, Plácido; Willner, Helge; Argüello, Gustavo A

    2005-08-01

    The trioxide, CF(3)OC(O)OOOC(O)OCF(3), reacts with NO(2) at 0 degrees C to yield the new peroxynitrate, CF(3)OC(O)OONO(2), which is stable for hours at room temperature. It is spectroscopically characterized and some thermal properties are reported. From the vapor pressure, ln(p/p(0)) = 14.06 - 4565/T, of the liquid above the melting point of -89 degrees C, the extrapolated boiling point is 52 degrees C. CF(3)OC(O)OONO(2) dissociates at higher temperatures and low pressures into the radicals CF(3)OC(O)OO and NO(2) as demonstrated by matrix isolation experiments. The matrix-isolated peroxy radicals consist in a rotameric mixture of trans,trans,trans-CF(3)OC(O)OO and trans,trans,cis-CF(3)OC(O)OO, where trans and cis denote dihedral angles of ca. 180 degrees and 0 degree, respectively, around beta F-C-O-C, beta C-O-C-O, and beta O-C-O-O, with an equilibrium composition dependent on the thermolysis temperature. The radical trans,trans,cis-CF(3)OC(O)OO is found to be ca. 3 kJ mol(-1) higher in enthalpy than trans,trans,trans-CF(3)OC(O)OO. DFT calculations are performed to support the vibrational assignments and to provide structural information about CF(3)OC(O)OONO(2). PMID:16060622

  17. Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates.

    PubMed

    Friman, V-P; Soanes-Brown, D; Sierocinski, P; Molin, S; Johansen, H K; Merabishvili, M; Pirnay, J-P; De Vos, D; Buckling, A

    2016-01-01

    Recent years have seen renewed interest in phage therapy--the use of viruses to specifically kill disease-causing bacteria--because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre-adapted all phage strains against all bacterial strains and then compared the efficacy of pre-adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre-adaptation, and as a result, phage therapies might need to be individually adjusted for different patients. PMID:26476097

  18. Characterization of the new metallo-beta-lactamase VIM-13 and its integron-borne gene from a Pseudomonas aeruginosa clinical isolate in Spain.

    PubMed

    Juan, Carlos; Beceiro, Alejandro; Gutiérrez, Olivia; Albertí, Sebastián; Garau, Margalida; Pérez, José L; Bou, Germán; Oliver, Antonio

    2008-10-01

    During a survey conducted to evaluate the incidence of class B carbapenemase (metallo-beta-lactamase [MBL])-producing Pseudomonas aeruginosa strains from hospitals in Majorca, Spain, five clinical isolates showed a positive Etest MBL screening test result. In one of them, strain PA-SL2, the presence of a new bla(VIM) derivative (bla(VIM-13)) was detected by PCR amplification with bla(VIM-1)-specific primers followed by sequencing. The bla(VIM-13)-producing isolate showed resistance to all beta-lactams (except aztreonam), gentamicin, tobramycin, and ciprofloxacin. VIM-13 exhibited 93% and 88% amino acid sequence identities with VIM-1 and VIM-2, respectively. bla(VIM-13) was cloned in parallel with bla(VIM-1), and the resistance profile conferred was analyzed both in Escherichia coli and in P. aeruginosa backgrounds. Compared to VIM-1, VIM-13 conferred slightly higher levels of resistance to piperacillin and lower levels of resistance to ceftazidime and cefepime. VIM-13 and VIM-1 were purified in parallel as well, and their kinetic parameters were compared. The k(cat)/K(m) ratios for the antibiotics mentioned above were in good agreement with the MIC data. Furthermore, EDTA inhibited the activity of VIM-13 approximately 25 times less than it inhibited the activity of VIM-1. VIM-13 was harbored in a class 1 integron, along with a new variant (Ala108Thr) of the aminoglycoside-modifying enzyme encoding gene aacA4, which confers resistance to gentamicin and tobramycin. Finally, the VIM-13 integron was apparently located in the chromosome, since transformation and conjugation experiments consistently yielded negative results and the bla(VIM-13) probe hybridized only with the genomic DNA. PMID:18644957

  19. A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

    PubMed Central

    Chen, Feifei; Xia, Yongjie; Lou, Jingyu; Zhang, Xue; Yang, Nana; Sun, Xiaoxu; Zhang, Qin; Zhuo, Chao; Huang, Xi; Deng, Xin; Yang, Cai-Guang; Ye, Yan; Zhao, Jing; Wu, Min; Lan, Lefu

    2014-01-01

    The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmSL181P, BfmSL181P/E376Q, and BfmSR393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs. PMID:25166864

  20. 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

  1. Genome-wide patterns of recombination in the opportunistic human pathogen Pseudomonas aeruginosa.

    PubMed

    Dettman, Jeremy R; Rodrigue, Nicolas; Kassen, Rees

    2015-01-01

    The bacterium Pseudomonas aeruginosa is a significant cause of acute nosocomial infections as well as chronic respiratory infections in patients with cystic fibrosis (CF). Recent reports of the intercontinental spread of a CF-specific epidemic strain, combined with high intrinsic levels of antibiotic resistance, have made this opportunistic pathogen an important public health concern. Strain-specific differences correlate with variation in clinical outcomes of infected CF patients, increasing the urgency to understand the evolutionary origin of genetic factors conferring important phenotypes that enable infection, virulence, or resistance. Here, we describe the genome-wide patterns of homologous and nonhomologous recombination in P. aeruginosa, and the extent to which the genomes are affected by these diversity-generating processes. Based on whole-genome sequence data from 32 clinical isolates of P. aeruginosa, we examined the rate and distribution of recombination along the genome, and its effect on the reconstruction of phylogenetic relationships. Multiple lines of evidence suggested that recombination was common and usually involves short stretches of DNA (200-300 bp). Although mutation was the main source of nucleotide diversity, the import of polymorphisms by homologous recombination contributed nearly as much. We also identified the genomic regions with frequent recombination, and the specific sequences of recombinant origin within epidemic strains. The functional characteristics of the genes contained therein were examined for potential associations with a pathogenic lifestyle or adaptation to the CF lung environment. A common link between many of the high-recombination genes was their functional affiliation with the cell wall, suggesting that the products of recombination may be maintained by selection for variation in cell-surface molecules that allows for evasion of the host immune system. PMID:25480685

  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. Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

    DOE PAGESBeta

    Keravec, Marlène; Mounier, Jérôme; Prestat, Emmanuel; Vallet, Sophie; Jansson, Janet K.; Burgaud, Gaëtan; Rosec, Sylvain; Gouriou, Stéphanie; Rault, Gilles; Coton, Emmanuel; et al

    2015-08-09

    Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly moremore » prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.« less

  4. Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

    SciTech Connect

    Keravec, Marlene; Mounier, Jerome; Prestat , Emmanuel; Vallet, Sophie; Jansson, Janet K.; Bergaud , Gaetaqn; Rosec, Silvain; Gourious, Stephanie; Rault, Gilles; Coton, Emmanuel; Barbier, George; Hery-Arnaud, Geneveieve

    2015-08-09

    Abstract Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly more prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.

  5. Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization.

    PubMed

    Keravec, Marlène; Mounier, Jérôme; Prestat, Emmanuel; Vallet, Sophie; Jansson, Janet K; Burgaud, Gaëtan; Rosec, Sylvain; Gouriou, Stéphanie; Rault, Gilles; Coton, Emmanuel; Barbier, Georges; Héry-Arnaud, Geneviève

    2015-01-01

    Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly more prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state. PMID:26266076

  6. Decomposition of dissolved organic matter released by an isolate of Microcystis aeruginosa and morphological profile of the associated bacterial community.

    PubMed

    Moreira, I C; Bianchini Jr, I; Vieira, A A H

    2011-02-01

    This study concerns the kinetics of bacterial degradation of two fractions (molecular mass) of dissolved organic matter (DOM) released by Microcystis aeruginosa. Barra Bonita Reservoir (SP, Brazil) conditions were simulated in the laboratory using the associated local bacterial community. The extent of degradation was quantified as the amount of organic carbon transferred from each DOM fraction (< 3 kDa and 3-30 kDa) to bacteria. The variation of bacteria morphotypes associated with the decomposition of each fraction was observed. To find the degradation rate constants (kT), the time profiles of the total, dissolved and particulate organic carbon concentrations were fitted to a first-order kinetic model. These rate constants were higher for the 3-30 kDa fraction than for the lighter fraction. Only in the latter fraction the formation of refractory dissolved organic carbon (DOCR) compounds could be detected and its rate of mass loss was low. The higher bacterial density was reached at 24 and 48 hours for small and higher fractions, respectively. In the first 48 hours of decomposition of both fractions, there was an early predominance of bacillus, succeeded by coccobacillus, vibrios and coccus, and from day 5 to 27, the bacterial density declined and there was greater evenness among the morphotypes. Both fractions of DOM were consumed rapidly, corroborating the hypothesis that DOM is readily available in the environment. This also suggests that the bacterial community in the inocula readily uses the labile part of the DOM, until this community is able to metabolise efficiently the remaining of DOM not degraded in the first moment. Given that M. aeruginosa blooms recur throughout the year in some eutrophic reservoirs, there is a constant supply of the same DOM which could maintain a consortium of bacterial morphotypes adapted to consuming this substrate. PMID:21437399

  7. Kinetics of nutrient enhanced crude oil degradation by Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2 isolated from Guwahati refinery, India.

    PubMed

    Chettri, Bobby; Mukherjee, Arghya; Langpoklakpam, James S; Chattopadhyay, Dhrubajyoti; Singh, Arvind K

    2016-09-01

    Bacterial degradation of crude oil in response to nutrient treatments has been vastly studied. But there is a paucity of information on kinetic parameters of crude oil degradation. Here we report the nutrient stimulated kinetic parameters of crude oil degradation assessed in terms of CO2 production and oil removal by Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2. The hydrocarbon degradation rate of P. aeruginosa AKS1 in oil only amended sediment was 10.75 ± 0.65 μg CO2-C g(-1) sediment day(-1) which was similar to degradation rate in sediments with no oil. In presence of both inorganic N & P, the degradation rate increased to 47.22 ± 1.32 μg CO2-C g(-1) sediment day(-1). The half-saturation constant (Ks) and maximum degradation rate (Vmax) for P. aeruginosa AKS1 under increasing N and saturating P concentration were 13.57 ± 0.53 μg N g(-1) sediment and 39.36 ± 1.42 μg CO2-C g(-1) sediment day(-1) respectively. The corresponding values at increasing P and a constant N concentration were 1.60 ± 0.13 μg P g(-1) sediment and 43.90 ± 1.03 μg CO2-C g(-1) sediment day(-1) respectively. Similarly the degradation rate of Bacillus sp. AKS2 in sediments amended with both inorganic nutrients N & P was seven fold higher than the rates in oil only or nutrient only treated sediments. The Ks and Vmax estimates of Bacillus sp. AKS2 under increasing N and saturating P concentration were 9.96 ± 1.25 μg N g(-1) sediment and 59.96 ± 7.56 μg CO2-C g(-1) sediment day(-1) respectively. The corresponding values for P at saturating N concentration were 0.46 ± 0.24 μg P g(-1) sediment and 63.63 ± 3.54 μg CO2-C g(-1) sediment day(-1) respectively. The rates of CO2 production by both isolates were further stimulated when oil concentration was increased above 12.5 mg g(-1) sediment. However, oil degradation activity declined at oil concentration above 40 mg g(-1) sediment when treated with constant nutrient: oil ratio

  8. Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches.

    PubMed

    Lund-Palau, Helena; Turnbull, Andrew R; Bush, Andrew; Bardin, Emmanuelle; Cameron, Loren; Soren, Odel; Wierre-Gore, Natasha; Alton, Eric W F W; Bundy, Jacob G; Connett, Gary; Faust, Saul N; Filloux, Alain; Freemont, Paul; Jones, Andy; Khoo, Valerie; Morales, Sandra; Murphy, Ronan; Pabary, Rishi; Simbo, Ameze; Schelenz, Silke; Takats, Zoltan; Webb, Jeremy; Williams, Huw D; Davies, Jane C

    2016-06-01

    Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development. PMID:27175979

  9. Classification of OprD sequence and correlation with antimicrobial activity of carbapenem agents in Pseudomonas aeruginosa clinical isolates collected in Japan.

    PubMed

    Sanbongi, Yumiko; Shimizu, Atsuyuki; Suzuki, Takahisa; Nagaso, Hiroshi; Ida, Takashi; Maebashi, Kazunori; Gotoh, Naomasa

    2009-07-01

    A total of 99 clinical isolates of metallo-ss-lactamase-negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so-called 'full length type', whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so-called 'defective type', which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in 'defective type' strains led to 15-, 17- and 23-fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with 'full length type' strains, respectively. 'Full length type' strains were further classified into six carbapenem susceptible types with the exception of four carbapenem-resistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, 'defective type' strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C-terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against 'full length type' strains were normal, whereas those against 'defective type' strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa. PMID:19563394

  10. Pseudomonas aeruginosa Can Be Detected in a Polymicrobial Competition Model Using Impedance Spectroscopy with a Novel Biosensor

    PubMed Central

    Ward, Andrew C.; Connolly, Patricia; Tucker, Nicholas P.

    2014-01-01

    Electrochemical Impedance Spectroscopy (EIS) is a powerful technique that can be used to elicit information about an electrode interface. In this article, we highlight six principal processes by which the presence of microorganisms can affect impedance and show how one of these - the production of electroactive metabolites - changes the impedance signature of culture media containing Pseudomonas aeruginosa. EIS, was used in conjunction with a low cost screen printed carbon sensor to detect the presence of P. aeruginosa when grown in isolation or as part of a polymicrobial infection with Staphylococcus aureus. By comparing the electrode to a starting measurement, we were able to identify an impedance signature characteristic of P. aeruginosa. Furthermore, we are able to show that one of the changes in the impedance signature is due to pyocyanin and associated phenazine compounds. The findings of this study indicate that it might be possible to develop a low cost sensor for the detection of P. aeruginosa in important point of care diagnostic applications. In particular, we suggest that a development of the device described here could be used in a polymicrobial clinical sample such as sputum from a CF patient to detect P. aeruginosa. PMID:24614411

  11. Antibiotic Resistance Pattern and Evaluation of Metallo-Beta Lactamase Genes Including bla-IMP and bla-VIM Types in Pseudomonas aeruginosa Isolated from Patients in Tehran Hospitals

    PubMed Central

    Aghamiri, Samira; Amirmozafari, Nour; Fallah Mehrabadi, Jalil; Fouladtan, Babak; Samadi Kafil, Hossein

    2014-01-01

    Beta-lactamase producing strains of Pseudomonas aeruginosa are important etiological agents of hospital infections. Carbapenems are among the most effective antibiotics used against Pseudomonas infections, but they can be rendered infective by group B β-lactamase, commonly called metallo-beta lactamase. In this study, the antimicrobial sensitivity patterns of P. aeruginosa strains isolated from 9 different hospitals in Tehran, Iran, as well as the prevalence of MBLs genes (bla-VIM and bla-IMP) were determined. A total of 212 strains of P. aeruginosa recovered from patients in hospitals in Tehran were confirmed by both biochemical methods and PCR. Their antimicrobial sensitivity patterns were determined by Kirby-Bauer disk diffusion method. Following MIC determination, imipenem resistant strains were selected by DDST method which was followed by PCR tests for determination of MBLs genes: bla-IMP and bla-VIM. The results indicated that, in the DDST phenotypic method, among the 100 imipenem resistant isolates, 75 strains were MBLs positive. The PCR test indicated that 70 strains (33%) carried bla-VIM gene and 20 strains (9%) harbored bla-IMP. The results indicated that the extent of antibiotic resistance among Pseudomonas aeruginosa is on the rise. This may be due to production of MBLs enzymes. Therefore, determination of antibiotic sensitivity patterns and MBLs production by these bacteria, can be important in control of clinical Pseudomonas infection. PMID:24944839

  12. Detection of blaSPM-1, blaKPC, blaTEM and blaCTX-M genes in isolates of Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella spp. from cancer patients with healthcare-associated infections.

    PubMed

    Jácome, Paula Regina Luna de Araújo; Alves, Lílian Rodrigues; Jácome-Júnior, Agenor Tavares; Silva, Maria Jesuíta Bezerra da; Lima, Jailton Lobo da Costa; Araújo, Paulo Sérgio Ramos; Lopes, Ana Catarina S; Maciel, Maria Amélia Vieira

    2016-07-01

    Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella spp. are three of the pathogens most frequently involved in infections of cancer patients, and the production of β -lactamases is a major mechanism of resistance due to its wide diversity of existing enzymes. Therefore, the aim of the present study was to investigate the microbiological profile and data related to patients and infections, and to search for β -lactamase genes in bacterial isolates from hospitalized cancer patients in a hospital in Recife, Pernambuco, Brazil. A total of 169 isolates were recovered between 2012 and 2014, of which 58 were P. aeruginosa, 36 were Acinetobacter spp. and 75 were Klebsiella spp. A high percentage of carbapenem resistance was observed in P. aeruginosa and Acinetobacter spp. Among the carbapenem-resistant bacteria, the blaSPM-1 gene was detected in P. aeruginosa (35.5 %) and Acinetobacter spp. (3.8 %), while blaKPC was detected in P. aeruginosa (25.8 %) only. Among the third- and fourth-generation cephalosporin-resistant strains, in Klebsiella spp. we detected the genes blaTEM (30.6 %), blaCTX-M (58.3 %) and blaKPC (5.6 %), and in Acinetobacter spp. only blaTEM (25.9 %). This the first report of an Acinetobacter baumannii blaSPM-1 gene carrier that has been isolated in Brazil. The most frequent cancer types were bowel tumour [14.8 %; 95 % confidence interval (CI95 %) 9.8-21.1 %], breast cancer (13.6 %; CI95 % 8.8-19.7 %) and prostate cancer (11.2%; CI95 % 6.9-17.0 %). These results therefore provide knowledge of susceptibility profile and resistance mechanisms and thus can contribute to the strategic formulation of hospital infection control plans and the rational use of antimicrobials, reducing mortality from infection levels in cancer patients. PMID:27217349

  13. Evolutionary insight from whole-genome sequencing of Pseudomonas aeruginosa from cystic fibrosis patients.

    PubMed

    Marvig, Rasmus Lykke; Sommer, Lea M; Jelsbak, Lars; Molin, Søren; Johansen, Helle Krogh

    2015-01-01

    The opportunistic pathogen Pseudomonas aeruginosa causes chronic airway infections in patients with cystic fibrosis (CF), and it is directly associated with the morbidity and mortality connected with this disease. The ability of P. aeruginosa to establish chronic infections in CF patients is suggested to be due to the large genetic repertoire of P. aeruginosa and its ability to genetically adapt to the host environment. Here, we review the recent work that has applied whole-genome sequencing to understand P. aeruginosa population genomics, within-host microevolution and diversity, mutational mechanisms, genetic adaptation and transmission events. Finally, we summarize the advances in relation to medical applications and laboratory evolution experiments. PMID:25865196

  14. Modeling The CF4 Laser

    NASA Astrophysics Data System (ADS)

    Patterson, C. W.; McDowell, R. S.; Krohn, B. J.; Nereson, N. G.

    1981-12-01

    The CO2-pumped CF4 laser is a potentially useful source of line-tunable infrared radiation in the region 605-655 cm 1, and the spectroscopy of CF4 has been carried to the point that the laser frequencies that will result from any given pump line can be calculated to better than 0.01 cm 1. We now report quantitative intensity and line-broadening studies on CF4 and their application to modeling the laser gain. First, absorption measurements on isolated lines in the ν2 + ν4 pump band at a series of pressures yield an effective transition dipole moment for this band of 0.010 Debye. At the same time the transition moment for the (ν2 + ν4) - ν2 laser band has been calculated and agrees well with the results of laser self-absorption measurements. Finally, linewidths determined as a function of pressure yield a pressure-broadening coefficient of ca. 10 MHz/torr, significantly greater than that expected from a hard-sphere gas-kinetic model. From these data the gain of the CF4 laser can be calculated at various pressures and temperatures; the results are in reasonable agreement with measured values.

  15. Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections

    PubMed Central

    Cigana, Cristina; Lorè, Nicola Ivan; Riva, Camilla; De Fino, Ida; Spagnuolo, Lorenza; Sipione, Barbara; Rossi, Giacomo; Nonis, Alessandro; Cabrini, Giulio; Bragonzi, Alessandra

    2016-01-01

    Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies. PMID:26883959

  16. Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections.

    PubMed

    Cigana, Cristina; Lorè, Nicola Ivan; Riva, Camilla; De Fino, Ida; Spagnuolo, Lorenza; Sipione, Barbara; Rossi, Giacomo; Nonis, Alessandro; Cabrini, Giulio; Bragonzi, Alessandra

    2016-01-01

    Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies. PMID:26883959

  17. Could the DiversiLab® semi-automated repetitive-sequence-based PCR be an acceptable technique for typing isolates of Pseudomonas aeruginosa? An answer from our experience and a review of the literature.

    PubMed

    Brossier, Florence; Micaelo, Maïté; Luyt, Charles-Edouard; Lu, Qin; Chastre, Jean; Arbelot, Charlotte; Trouillet, Jean-Louis; Combes, Alain; Rouby, Jean-Jacques; Jarlier, Vincent; Aubry, Alexandra

    2015-12-01

    Recently the DiversiLab® (DL) system (bioMérieux) was developed as an automated platform that uses repetitive element polymerase chain reaction (rep-PCR) technology for standardized, reproducible DNA fingerprinting of bacteria. The purpose of this study was to evaluate the usefulness of DL rep-PCR for typing Pseudomonas aeruginosa isolates. The performance of DL rep-PCR was compared with that of pulsed-field gel electrophoresis (PFGE) in a prospective multicenter study of patients with ventilator-associated pneumonia due to P. aeruginosa, conducted in 3 intensive care units over a 31-month period. In total, 203 P. aeruginosa isolates from 66 patients, from whom at least 2 consecutive respiratory samples each were collected more than 48 h apart, were typed using DL rep-PCR. Forty isolates (corresponding to 20 patients) were also typed using PFGE of SpeI-digested DNA. The typeability was 100% with DL rep-PCR and 95% with PFGE. The discriminatory power was close for DL rep-PCR and for PFGE (Simpson's diversity indices of 0.901 and 0.947, respectively). Insufficient agreement between DL rep-PCR and PFGE typing results was observed for the 40 selected isolates (adjusted Rand coefficient of 0.419), mostly due to isolates of the same DL rep-PCR type but of different PFGE types (adjusted Wallace coefficients of 0.306 for DL rep-PCR with PFGE, and of 0.667 for PFGE with DL rep-PCR). Considered together with published data, DL rep-PCR results should be interpreted with caution for the investigation of outbreaks caused by P. aeruginosa and evaluated in conjunction with epidemiological data. PMID:26460809

  18. Study on imipenem resistance and prevalence of blaVIM1 and blaVIM2 metallo-beta lactamases among clinical isolates of Pseudomonas aeruginosa from Mashhad, Northeast of Iran

    PubMed Central

    Mirbagheri, Seyedeh Zohreh; Meshkat, Zahra; Naderinasab, Mahboubeh; Rostami, Sina; Nabavinia, Maryam Sadat; Rahmati, Mehdi

    2015-01-01

    Background and Objectives: The main cause of serious nosocomial infections is a Gram-negative pathogen known as Pseudomonas aeruginosa (P. aeruginosa). Carbapenems are widely used as an appropriate treatment for these infections, however resistance to these agents has been observed and is increasing. Metallo beta-lactamase (MBLs) enzyme is one of the main causes of resistance to carbapenem. In the current study the frequency and production of VIM1 and VIM2 by imipenem-resistant P. aeruginosa isolates of patients hospitalized in Imam Reza hospital were evaluated. Materials and Methods: In this study, 131 clinical samples were collected from patients hospitalized in Imam Reza hospital in Mashhad during a 15-month period from May 2011 to November 2012. After verification of P. aeruginosa isolates, antibiotic resistance patterns of isolates were determined for 14 antibiotics by Kirby-Bauer standard disk diffusion according to the CLSI guidelines. Combined-disk test was used for phenotypic determination of MBLs-producing isolates and after DNA extraction, genotypic determination of VIM1 and VIM2 metallo beta-lactamase genes was carried out using Multiplex-PCR. Results: Of 63 imipenem-resistant isolates (48.5%), 56 (88.8%) were MBL-producing in phenotypic assessments. Also amongst imipenem-resistant isolates, the frequency of VIM1 and VIM2 genes were 58.7 and 3.17%, respectively. Conclusion: The results of the current study along with the results of the other conducted studies in Iran in recent years demonstrate that the average resistance to imipenem in P. aeruginosa isolates was 51.3% which has increased in comparison with the results in 2006 (32.9%). It was also determined that the frequency of VIM1 gene was more than VIM2 gene. In phenotypic assessment by using CD method, 49.6% of isolates were determined as MBLs-producing. The sensitivity and specificity of this method were verified in comparison with the results of PCR test. PMID:26622967

  19. [Carriage of class 1 and 2 integrons in Acinetobacter baumannii and Pseudomonas aeruginosa isolated from clinical specimens and a novel gene cassette array: blaOXA-11-cmlA7].

    PubMed

    Mengeloğlu, Fırat Zafer; Copur Çiçek, Ayşegül; Koçoğlu, Esra; Sandallı, Cemal; Budak, Emine Esra; Ozgümüş, Osman Birol

    2014-01-01

    The dissemination of antibiotic resistance genes between bacteria leads to serious problems in the treatment of infectious diseases. It has been shown that resistance genes can also be carried by the integrons. There are limited studies regarding the carriage of class 1 and 2 integrons in Acinetobacter baumannii and Pseudomonas aeruginosa clinical strains in Turkey. The aims of this study were to investigate the carriage rates of class 1 and class 2 integrons in A.baumannii and P.aeruginosa strains isolated from clinical samples in Abant Izzet Baysal University Hospital, and to characterize the antibiotic resistance gene cassettes in these integrons by sequence analyses. A total of 137 strains (77 A.baumannii and 60 P.aeruginosa) isolated from various clinical specimens (56% were sputum, 19% wound, 11% urine, 11% blood, 3% catheter), between March 2010-December 2012, were included in the study. The identification and antibiotic susceptibility tests of the isolates were performed by Vitek 2 Compact (bioMérieux, France) and BD Phoenix 100 (Becton Dickinson, USA) systems. The presence of integrons were screened by PCR method using specific primer pairs targeting class 1 (intI1) and 2 (intI2) integrase regions. All the samples that revealed integron amplification were subjected to DNA sequence analysis, both in the forms of cloned products and PCR amplicons. In the study, the highest susceptibility rates were found against colistin (96%) and tigecycline (78%) in A.baumannii, and against piperacillin/tazobactam (97%) and piperacillin (93%) in P.aeruginosa isolates. The highest resistance rate was determined for piperacillin/tazobactam (95%) in A.baumannii strains. The presence of intI1 gene was detected in 33% (26/77) of A.baumannii and 10% (6/60) of P.aeruginosa isolates. When variable regions in intI1 positive strains were amplified by PCR, eight (8/77, 10%) A.baumannii and three (3/60, 5%) P.aeruginosa strains were found to harbor antibiotic resistance gene

  20. The effects of D-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa.

    PubMed

    She, Pengfei; Chen, Lihua; Liu, Hongbo; Zou, Yaru; Luo, Zhen; Koronfel, Asmaa; Wu, Yong

    2015-09-01

    The biofilm formation of microorganisms causes persistent tissue infections resistant to treatment with antimicrobial agents. Pseudomonas aeruginosa is commonly isolated from the airways of patients with chronic fibrosis (CF) and often forms biofilms, which are extremely hard to eradicate and a major cause of mortality and morbidity. Recent studies have shown that D-amino acids (D-AAs) inhibited and disrupted biofilm formation by causing the release of the protein component of the polymeric matrix. However, the effects of D-AAs combined with common antibiotics on biofilms have rarely been studied. The current study first determined whether D-AAs disrupted the biofilms of PAO1 and the clinical airway isolates of P. aeruginosa. It was then determined whether combinations of D-Tyr (the most effective one) and the antibiotic amikacin (AMK) enhanced the activity against these biofilms. The results of the current study showed that D-Tyr is the most effective among those that disassemble the D-amino acids (D-leucine, D-methionine, D-Tyrptophan, and D-tryptophan), and D-Tyr at concentrations higher than 5 mM significantly reduced the biofilm biomass of P. aeruginosa (p < 0.05) without influencing bacterial growth. It was also revealed that D-Tyr improved the efficacy of AMK to combat P. aeruginosa biofilms, as indicated by a reduction in the minimal biofilm-inhibiting concentration (MBIC50 and MBIC90) without a change in the minimal inhibitory concentration (MIC) of planktonic bacteria. Thus, the findings indicated that D-Tyr supplementation overcame the resistance of P. aeruginosa biofilms to AMK, which might be helpful for preventing AMK overuse when this specific D-Tyr is recommended for combatting these biofilms. Also, toxicity of the liver and kidney from AMK could be potentially mitigated by co-delivery with D-Tyr. PMID:26188263

  1. Assessment of the Microbial Constituents of the Home Environment of Individuals with Cystic Fibrosis (CF) and Their Association with Lower Airways Infections

    PubMed Central

    Heirali, Alya; McKeon, Suzanne; Purighalla, Swathi; Storey, Douglas G.; Rossi, Laura; Costilhes, Geoffrey; Drews, Steven J.; Rabin, Harvey R.; Surette, Michael G.; Parkins, Michael D.

    2016-01-01

    Introduction Cystic fibrosis (CF) airways are colonized by a polymicrobial community of organisms, termed the CF microbiota. We sought to define the microbial constituents of the home environment of individuals with CF and determine if it may serve as a latent reservoir for infection. Methods Six patients with newly identified CF pathogens were included. An investigator collected repeat sputum and multiple environmental samples from their homes. Bacteria were cultured under both aerobic and anaerobic conditions. Morphologically distinct colonies were selected, purified and identified to the genus and species level through 16S rRNA gene sequencing. When concordant organisms were identified in sputum and environment, pulsed-field gel electrophoresis (PFGE) was performed to determine relatedness. Culture-independent bacterial profiling of each sample was carried out by Illumina sequencing of the V3 region of the 16s RNA gene. Results New respiratory pathogens prompting investigation included: Mycobacterium abscessus(2), Stenotrophomonas maltophilia(3), Pseudomonas aeruginosa(3), Pseudomonas fluorescens(1), Nocardia spp.(1), and Achromobacter xylosoxidans(1). A median 25 organisms/patient were cultured from sputum. A median 125 organisms/home were cultured from environmental sites. Several organisms commonly found in the CF lung microbiome were identified within the home environments of these patients. Concordant species included members of the following genera: Brevibacterium(1), Microbacterium(1), Staphylococcus(3), Stenotrophomonas(2), Streptococcus(2), Sphingomonas(1), and Pseudomonas(4). PFGE confirmed related strains (one episode each of Sphinogomonas and P. aeruginosa) from the environment and airways were identified in two patients. Culture-independent assessment confirmed that many organisms were not identified using culture-dependent techniques. Conclusions Members of the CF microbiota can be found as constituents of the home environment in individuals with

  2. Pseudomonas aeruginosa uses multiple pathways to acquire iron during chronic infection in cystic fibrosis lungs.

    PubMed

    Konings, Anna F; Martin, Lois W; Sharples, Katrina J; Roddam, Louise F; Latham, Roger; Reid, David W; Lamont, Iain L

    2013-08-01

    Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription-quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF. PMID:23690396

  3. 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

  4. 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

  5. Modeling the CF4 laser

    NASA Astrophysics Data System (ADS)

    Patterson, C. W.; McDowell, R. S.; Krohn, B. J.; Nereson, N. G.

    Quantitative intensity and line broadening studies on CF4 and their application to modeling the laser gain is presented. First, absorption measurements on isolated lines in the v sub 2 + v sub 4 pump band at a series of pressures yield an effective transition dipole moment for this band of 0.010 Debye. At the same time the transition moment for the v sub 2 + v sub 4 - v sub 2 laser band are calculated and agrees well with the results of laser self-absorption measurements. Finally, linewidths determined as a function of pressure yield a pressure broadening coefficient of ca, 10 MHz/torr, significantly greater than that expected from a hard sphere gas kinetic model. From these data the gain of the CF4 laser are calculated at various pressures and temperatures; the results are in reasonable agreement with measured values.

  6. 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

  7. Differences in antimicrobial susceptibility breakpoints for Pseudomonas aeruginosa, isolated from blood cultures, set by the Clinical and Laboratory Standards Institute (CLSI) and the Japanese Society of Chemotherapy.

    PubMed

    Nakamura, Tatsuya; Shimizu, Chihiro; Kasahara, Mayumi; Nakata, Chiyo; Munakata, Machiko; Takahashi, Hakuo

    2007-02-01

    A study was made of the antimicrobial susceptibility to and efficacy of various kinds of antimicrobial agents against 179 strains of Pseudomonas aeruginosa that were isolated from blood cultures at Kansai Medical University Hospital from 1990 through 2004. The annual detection rate was highest in 1994, at 22 strains (6.5%). There were 9 multidrug resistant strains of Pseudomonas aeruginosa (5.0%). Among 14 antimicrobial agents tested for measurements, ciprofloxacin (CPFX) showed the best minimum inhibitory concentration (MIC) 50 value, of 0.25 microg/ml, followed by pazufloxacin (PZFX) and biapenem (BIPM), each at 0.5 microg/ml. When the period of 15 years was divided into three stages, the MIC50 value for each antimicrobial agent was highest in the middle stage (1995 to 1999). Assuming that the percentage of sensitive strains according to the breakpoints set by the Clinical and Laboratory Standards Institute (CLSI) represents the antimicrobial susceptibility rate, amikacin (AMK) showed the best value, of 85.5%. According to the sepsis breakpoint set by the Japanese Society of Chemotherapy (JSC), the efficacy of CPFX showed the highest rate (77.1%) of all the antimicrobial agents tested. Among beta-lactams, BIPM showed the highest efficacy rate, of 67.0%. When the efficacy rates were compared with each other, the difference in efficacy rate between the breakpoint set by the CLSI and the sepsis breakpoint set by the JSC was large for beta-lactams. Comparisons made based on the CLSI criteria showed no difference in cross-resistance rates between CPFX, meropenem (MEPM), and BIPM. However, when comparisons were made using the JSC sepsis breakpoint, MEPM showed a cross-resistance rate of 87.8%, while the rate for BIPM was lower, at 56.1%, with the chi2 test showing a significant difference, at P = 0.0014. In accordance with the pharmacokinetics/pharmacodynamics theory that has been advocated, breakpoints which are more suitable for the clinical setting in Japan should

  8. OXA-46, a new class D beta-lactamase of narrow substrate specificity encoded by a blaVIM-1-containing integron from a Pseudomonas aeruginosa clinical isolate.

    PubMed

    Giuliani, Francesco; Docquier, Jean-Denis; Riccio, Maria Letizia; Pagani, Laura; Rossolini, Gian Maria

    2005-05-01

    A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a bla(VIM-1) metallo-beta-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D beta-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of bla(OXA-46) in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D beta-lactamases. PMID:15855521

  9. Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients

    PubMed Central

    2010-01-01

    Background Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains. Results All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness. Conclusions The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results. PMID:20374629

  10. 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

  11. 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

  12. Atmospheric chemistry of CF3CF2OCH3

    NASA Astrophysics Data System (ADS)

    Østerstrøm, Freja F.; Nielsen, Ole John; Wallington, Timothy J.

    2016-06-01

    Smog chamber Fourier transform infrared techniques were used to investigate the kinetics of the reaction of CF3CF2OCH3 with Cl atoms and OH radicals: k(Cl + CF3CF2OCH3) = (1.09 ± 0.16) × 10-13 and k(OH + CF3CF2OCH3) = (1.28 ± 0.19) × 10-14 cm3 molecule-1 s-1 in 700 Torr total pressure of N2/O2 at 296 ± 2 K. The Cl-initiated oxidation of CF3CF2OCH3 gives CF3CF2OCHO in a yield indistinguishable from 100%. An estimate of k(Cl + CF3CF2OCHO) = (1.18 ± 0.34) × 10-14 cm3 molecule-1 s-1 is provided. Based on the OH reaction rate, the atmospheric lifetime of CF3CF2OCH3 is estimated to be 5.0 years. The 100-year time horizon global warming potential of CF3CF2OCH3 is estimated to be 585. The atmospheric impact of CF3CF2OCH3 is discussed.

  13. VAPOR PRESSURES, LIQUID MOLAR VOLUMES, VAPOR NON- IDEALITY, AND CRITICAL PROPERTIES OF CF3OCF2CF2CF3, c-CF2CF2CF2CF2O, CF3OCF2OCF3, AND CF3OCF2CF2H

    EPA Science Inventory

    New measurements of the thermophysical properties of CF3OCF2CF2CF3 and c -CF2CF2CF2CF2O are reported from T ≈ 235 K to the critical region. Liquid-phase volumetric results for CF3OCF2OCF3 and CF3OCF2CF2H (235 < T/K < 303) are reported to supplement the information already availab...

  14. Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

    PubMed Central

    Luan, Xiaojie; Campanucci, Verónica A.; Nair, Manoj; Yilmaz, Orhan; Belev, George; Machen, Terry E.; Chapman, Dean; Ianowski, Juan P.

    2014-01-01

    Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation. PMID:25136096

  15. Clonal Dissemination, Emergence of Mutator Lineages and Antibiotic Resistance Evolution in Pseudomonas aeruginosa Cystic Fibrosis Chronic Lung Infection

    PubMed Central

    Mulet, Xavier; Cabot, Gabriel; Moyà, Bartolomé; Figuerola, Joan; Togores, Bernat; Pérez, José L.; Oliver, Antonio

    2013-01-01

    Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator

  16. Complete Sequence of pOZ176, a 500-Kilobase IncP-2 Plasmid Encoding IMP-9-Mediated Carbapenem Resistance, from Outbreak Isolate Pseudomonas aeruginosa 96

    PubMed Central

    Xiong, Jianhui; Alexander, David C.; Ma, Jennifer H.; Déraspe, Maxime; Low, Donald E.; Jamieson, Frances B.

    2013-01-01

    Pseudomonas aeruginosa 96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 of Pseudomonas fluorescens SBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. The blaIMP-9-carrying integron contained aacA4→blaIMP-9→aacA4, flanked upstream by Tn21 tnpMRA and downstream by a complete tni operon of Tn402 and a mer module, named Tn6016. The second integron carried aacA4→catB8a→blaOXA-10 and was flanked by Tn1403-like tnpRA and a sul1-type 3′ conserved sequence (3′-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and two pil operons. The replication and maintenance systems exhibit similarity to a genomic island of Ralstonia solanacearum GM1000. Codon usage analysis suggests the recent acquisition of blaIMP-9. The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria. PMID:23716048

  17. A study of the efficiency of edible oils degraded in alkaline conditions by Pseudomonas aeruginosa SS-219 and Acinetobacter sp. SS-192 bacteria isolated from Japanese soil.

    PubMed

    Sugimori, Daisuke; Utsue, Tomohiro

    2012-03-01

    High lipid concentration contained in wastewater inhibits the activity of microorganisms in biological wastewater treatment systems such as activated sludge and methane fermentation. To reduce the inhibitory effects, microorganisms capable of efficiently degrading edible oils were screened from various environmental sources. From Japanese soil, we isolated 2 bacteria strains with high degradation abilities at an alkaline pH without consumption of biological oxygen demand (BOD) constituents. Acinetobacter sp. strain SS-192 and Pseudomonas aeruginosa strain SS-219 degraded 77.5 ± 0.6% and 89.5 ± 1.5%, respectively, of 3,000 ppm of mixed oil consisting of salad oil/lard/beef tallow (1/1/1, w/w/w) at 37°C and pH 9.0 in 24 h. Efficient degradation by the two strains occurred at pH 8-9 and 25-40°C. Strain SS-219 degraded lipids even at pH 3. The degradation rate of 3,000 ppm of salad oil, lard, and beef tallow by strain SS-192 was 79.9 ± 2.6%, 63.6 ± 1.9%, and 70.1 ± 1.2%, respectively, during a 24-h cultivation. The degradation rate of 3,000 ppm of salad oil, lard, and beef tallow by strain SS-219 was 82.3 ± 2.1%, 71.9 ± 2.2%, and 71.0 ± 1.1%, respectively, during a 24-h cultivation. After mixed oil degradation by both strains, the BOD value of the cell culture increased from 2,100 ppm to 3,200-4,000 ppm. The fact that neither strain utilizes BOD ingredients will be beneficial to pretreatment of methane fermentation systems such as upflow anaerobic sludge blanket reactors. In addition, the growth of usual heterotrophic microorganisms utilizing soluble BOD can be suppressed under alkaline pH. PMID:22805803

  18. Structural characterization and surface activities of biogenic rhamnolipid surfactants from Pseudomonas aeruginosa isolate MN1 and synergistic effects against methicillin-resistant Staphylococcus aureus.

    PubMed

    Samadi, Nasrin; Abadian, Neda; Ahmadkhaniha, Reza; Amini, Farzaneh; Dalili, Dina; Rastkari, Noushin; Safaripour, Eliyeh; Mohseni, Farzaneh Aziz

    2012-11-01

    The aim of present work was to study chemical structures and biological activities of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa MN1 isolated from oil-contaminated soil. The results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that total rhamnolipids (RLs) contained 16 rhamnolipid homologues. Di-lipid RLs containing C(10)-C(10) moieties were by far the most predominant congeners among mono-rhamnose (53.29 %) and di-rhamnose (23.52 %) homologues. Mono-rhamnolipids form 68.35 % of the total congeners in the RLs. Two major fractions were revealed in the thin layer chromatogram of produced RLs which were then purified by column chromatography. The retardation factors (R (f)) of the two rhamnolipid purple spots were 0.71 for RL1 and 0.46 for RL2. LC-MS/MS analysis proved that RL1 was composed of mono-RLs and RL2 consisted of di-RLs. RL1 was more surface-active with the critical micelle concentration (CMC) value of 15 mg/L and the surface tension of 25 mN/m at CMC. The results of biological assay showed that RL1 is a more potent antibacterial agent than RL2. All methicillin-resistant Staphylococcus aureus (MRSA) strains were inhibited by RLs that were independent of their antibiotic susceptibility patterns. RLs remarkably enhanced the activity of oxacillin against MRSA strains and lowered the minimum inhibitory concentrations of oxacillin to the range of 3.12-6.25 μg/mL. PMID:22644668

  19. Cystic Fibrosis Transmembrane Conductance Regulator is an Epithelial Cell Receptor for Clearance of Pseudomonas aeruginosa from the Lung

    NASA Astrophysics Data System (ADS)

    Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.

    1997-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant Δ F508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

  20. Bis(trifluoroacetyl) peroxide, CF(3)C(O)OOC(O)CF(3).

    PubMed

    Kopitzky, R; Willner, H; Hermann, A; Oberhammer, H

    2001-06-01

    Pure, highly explosive CF(3)C(O)OOC(O)CF(3) is prepared for the first time by low-temperature reaction between CF(3)C(O)Cl and Na(2)O(2). At room temperature CF(3)C(O)OOC(O)CF(3) is stable for days in the liquid or gaseous state. The melting point is -37.5 degrees C, and the boiling point is extrapolated to 44 degrees C from the vapor pressure curve log p = -1875/T + 8.92 (p/mbar, T/K). Above room temperature the first-order unimolecular decay into C(2)F(6) + CO(2) occurs with an activation energy of 129 kJ mol(-1). CF(3)C(O)OOC(O)CF(3) is a clean source for CF(3) radicals as demonstrated by matrix-isolation experiments. The pure compound is characterized by NMR, vibrational, and UV spectroscopy. The geometric structure is determined by gas electron diffraction and quantum chemical calculations (HF, B3PW91, B3LYP, and MP2 with 6-31G basis sets). The molecule possesses syn-syn conformation (both C=O bonds synperiplanar to the O-O bond) with O-O = 1.426(10) A and dihedral angle phi(C-O-O-C) = 86.5(32) degrees. The density functional calculations reproduce the experimental structure very well. PMID:11375681

  1. Matrix isolation studies of the interactions of BF3 with water and substituted diethyl ethers. Chemical ionization mass spectrometric determination of the proton affinity of (CF3CH2)2O

    NASA Technical Reports Server (NTRS)

    Ball, David W.; Zehe, Michael J.

    1993-01-01

    BF3 was co-condensed with H2O, D2O, (C2H5)2O, (CF3CH2)2O, and (C2F5)2O in excess argon at 15 K. Infrared spectra of BF3/water isolated in solid argon provided a more complete analysis of the BF3--H2O complex than previously published. Infrared spectra of the matrices showed a definite Lewis acid-base interaction between BF3 and diethyl ether; a weak but definite interaction with bis (2,2,2-trifluorodiethyl) ether, and no observable interaction with perfluorodiethyl ether. Thus, the ether data indicate a clear trend between strength of interaction with BF3 and the degree of F substitution. To support and explain the emerging relationship between interaction strength and the basicity of the oxygen-containing molecule, the proton affinity of (CF3CH2)2O was measured using chemical ionization mass spectrometry. The implications of the results for lubricant/metal oxide surface interactions are discussed.

  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. Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum

    PubMed Central

    Line, Laura; Alhede, Morten; Kolpen, Mette; Kühl, Michael; Ciofu, Oana; Bjarnsholt, Thomas; Moser, Claus; Toyofuku, Masanori; Nomura, Nobuhiko; Høiby, Niels; Jensen, Peter Ø.

    2014-01-01

    Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO−3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO−3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 μM NO−3 and 100 μM NO−3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO−3. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO−3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO−3 followed by a transient increase of NO−2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P

  4. 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

  5. Inhalation with Fucose and Galactose for Treatment of Pseudomonas Aeruginosa in Cystic Fibrosis Patients

    PubMed Central

    Hauber, Hans-Peter; Schulz, Maria; Pforte, Almuth; Mack, Dietrich; Zabel, Peter; Schumacher, Udo

    2008-01-01

    Background: Colonisation of cystic fibrosis (CF) lungs with Pseudomonas aeruginosa is facilitated by two lectins, which bind to the sugar coat of the surface lining epithelia and stop the cilia beating. Objectives: We hypothesized that P. aeruginosa lung infection should be cleared by inhalation of fucose and galactose, which compete for the sugar binding site of the two lectins and thus inhibit the binding of P. aeruginosa. Methods: 11 adult CF patients with chronic infection with P. aeruginosa were treated twice daily with inhalation of a fucose/galactose solution for 21 days (4 patients only received inhalation, 7 patients received inhalation and intravenous antibiotics). Microbial counts of P. aeruginosa, lung function measurements, and inflammatory markers were determined before and after treatment. Results: The sugar inhalation was well tolerated and no adverse side effects were observed. Inhalation alone as well as combined therapy (inhalation and antibiotics) significantly decreased P. aeruginosa in sputum (P < 0.05). Both therapies also significantly reduced TNFα expression in sputum and peripheral blood cells (P < 0.05). No change in lung function measurements was observed. Conclusions: Inhalation of simple sugars is a safe and effective measure to reduce the P. aeruginosa counts in CF patients. This may provide an alternative therapeutical approach to treat infection with P. aeruginosa. PMID:19043609

  6. Transcriptional Activation of Mucin by Pseudomonas aeruginosa Lipopolysaccharide in the Pathogenesis of Cystic Fibrosis Lung Disease

    NASA Astrophysics Data System (ADS)

    Li, Jian-Dong; Dohrman, Austin F.; Gallup, Marianne; Miyata, Susumu; Gum, James R.; Kim, Young S.; Nadel, Jay A.; Prince, Alice; Basbaum, Carol B.

    1997-02-01

    An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a CI ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

  7. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

    PubMed Central

    Govan, J R; Deretic, V

    1996-01-01

    Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity. PMID:8840786

  8. 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

  9. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: rapid identification of bacteria isolated from patients with cystic fibrosis.

    PubMed

    Baillie, S; Ireland, K; Warwick, S; Wareham, D; Wilks, M

    2013-01-01

    Despite extensive research into the diagnosis and management of cystic fibrosis (CF) over the past decades, sufferers still have a median life expectancy of less than 37 years. Respiratory tract infections have a significant role in increasing the morbidity and mortality of patients with CF via a progressive decline in lung function. Rapid identification of organisms recovered from CF sputum is necessary for effective management of respiratory tract infections; however, standard techniques of identification are slow, technically demanding and expensive. The aim of this study is to asses the suitability of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) in identifying bacteria isolated from the respiratory tract of patients with CF, and is assessed by testing the accuracy of MALDI-TOF MS in identifying samples from a reference collection of rare CF strains in conjunction with comparing MALDI-TOF MS and standard techniques in identifying clinical isolates from sputum samples of CF patients. MALDI-TOF MS accurately identified 100% of isolates from the reference collection of rare CF pathogens (EuroCare CF collection). The isolate identification given by MALDI-TOF MS agreed with that given by standard techniques for 479/481 (99.6%) clinical isolates obtained from respiratory samples provided by patients with CE In two (0.4%) of 481 samples there was a discrepancy in identification between MALDI-TOF MS and standard techniques. One organism was identified as Pseudomonas aeruginosa by MALDI-TOF but could only be identified by the laboratory's standard methods as of the Pseudomonas genus. The second organism was identified as P. beteli by MALDI-TOF MS and Stenotrophomonas maltophilia by standard methods. This study shows that MALDI-TOF MS is superior to standard techniques in providing cheap, rapid and accurate identification of CF sputum isolates. PMID:24400425

  10. 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

  11. Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

    PubMed Central

    Abdullah, Lubna H.; Perlmutt, Olivia S.; Albert, Daniel; Davis, C. William; Arnold, Roland R.; Yankaskas, James R.; Gilligan, Peter; Neubauer, Heiner; Randell, Scott H.; Boucher, Richard C.

    2014-01-01

    The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages. PMID:25156735

  12. Introduction of Pseudomonas aeruginosa into a Hospital via Vegetables

    PubMed Central

    Kominos, Spyros D.; Copeland, Charles E.; Grosiak, Barbara; Postic, Bosko

    1972-01-01

    Pseudomonas aeruginosa was isolated from tomatoes, radishes, celery, carrots, endive, cabbage, cucumbers, onions, and lettuce obtained from the kitchen of a general hospital, with tomatoes yielding both highest frequencies of isolation and highest counts. Presence of P. aeruginosa on the hands of kitchen personnel and cutting boards and knives which they used suggests acquisition of the organism through contact with these vegetables. It is estimated that a patient consuming an average portion of tomato salad might ingest as many as 5 × 103 colony-forming units of P. aeruginosa. Pyocine types of P. aeruginosa isolated from clinical specimens were frequently identical to those recovered from vegetables, thus implicating tomatoes and other vegetables as an important source and vehicle by which P. aeruginosa colonizes the intestinal tract of patients. PMID:4628795

  13. CF6 performance improvement

    NASA Technical Reports Server (NTRS)

    Lennard, D. J.

    1978-01-01

    Potential CF6 engine performance improvements directed at reduced fuel consumption were identified and screened relative to airline acceptability and are reviewed. The screening process developed to provide evaluations of fuel savings and economic factors including return on investment and direct operating cost is described. In addition, assessments of development risk and production potential are made. Several promising concepts selected for full-scale development based on a ranking involving these factors are discussed.

  14. 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

  15. 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

  16. A new perfluorinated peroxynitrate, CF3CF2CF2CF2OONO2. Synthesis, characterization and atmospheric implications

    NASA Astrophysics Data System (ADS)

    Bossolasco, Adriana G.; Vila, Jesús A.; Burgos Paci, Maxi A.; Malanca, Fabio E.; Argüello, Gustavo A.

    2014-09-01

    CF3CF2CF2CF2OONO2 was synthesized from the photolysis of CF3CF2CF2CF2I, in presence of NO2 and O2. Alkyl peroxynitrates (CxF2x+1OONO2) could be formed in the atmospheric degradation of chlorofluorocarbons, hydrofluorocarbons and hydrofluoroethers. We present here the synthesis and characterization (IR and UV absorption cross sections) of CF3CF2CF2CF2OONO2 and its comparison with those corresponding to other perfluoro alkyl peroxynitrates. The thermal stability was studied as a function of total pressure (from 9.0 to 417 mbar) and temperature (from 283 to 293 K) using infrared spectroscopy. Kinetic parameters measured for the thermal dissociation were Ea = (81 ± 4) kJ/mol and A = 4.8 × 1012. DFT calculations at the B3LYP/6-311+G∗ level were used to explore the ground state potential energy surface. Geometrical parameters, conformer populations and vibrational spectra are presented. The calculated activation energy was 81.3 kJ mol-1 in excellent agreement with experimental results. Atmospheric implications are discussed.

  17. Distributed lags time series analysis versus linear correlation analysis (Pearson's r) in identifying the relationship between antipseudomonal antibiotic consumption and the susceptibility of Pseudomonas aeruginosa isolates in a single Intensive Care Unit of a tertiary hospital.

    PubMed

    Erdeljić, Viktorija; Francetić, Igor; Bošnjak, Zrinka; Budimir, Ana; Kalenić, Smilja; Bielen, Luka; Makar-Aušperger, Ksenija; Likić, Robert

    2011-05-01

    The relationship between antibiotic consumption and selection of resistant strains has been studied mainly by employing conventional statistical methods. A time delay in effect must be anticipated and this has rarely been taken into account in previous studies. Therefore, distributed lags time series analysis and simple linear correlation were compared in their ability to evaluate this relationship. Data on monthly antibiotic consumption for ciprofloxacin, piperacillin/tazobactam, carbapenems and cefepime as well as Pseudomonas aeruginosa susceptibility were retrospectively collected for the period April 2006 to July 2007. Using distributed lags analysis, a significant temporal relationship was identified between ciprofloxacin, meropenem and cefepime consumption and the resistance rates of P. aeruginosa isolates to these antibiotics. This effect was lagged for ciprofloxacin and cefepime [1 month (R=0.827, P=0.039) and 2 months (R=0.962, P=0.001), respectively] and was simultaneous for meropenem (lag 0, R=0.876, P=0.002). Furthermore, a significant concomitant effect of meropenem consumption on the appearance of multidrug-resistant P. aeruginosa strains (resistant to three or more representatives of classes of antibiotics) was identified (lag 0, R=0.992, P<0.001). This effect was not delayed and it was therefore identified both by distributed lags analysis and the Pearson's correlation coefficient. Correlation coefficient analysis was not able to identify relationships between antibiotic consumption and bacterial resistance when the effect was delayed. These results indicate that the use of diverse statistical methods can yield significantly different results, thus leading to the introduction of possibly inappropriate infection control measures. PMID:21277747

  18. Susceptibility of clinical isolates of Pseudomonas aeruginosa in the Northern Kyushu district of Japan to carbapenem antibiotics, determined by an integrated concentration method: evaluation of the method based on Monte Carlo simulation.

    PubMed

    Nagasawa, Zenzo; Kusaba, Koji; Aoki, Yosuke

    2008-06-01

    In empirical antibacterial therapy, regional surveillance is expected to yield important information for the determination of the class and dosage regimen of antibacterial agents to be used when dealing with infections with organisms such as Pseudomonas aeruginosa, in which strains resistant to antibacterial agents have been increasing. The minimal inhibitory concentrations (MICs) of five carbapenem antibiotics against P. aeruginosa strains isolated in the Northern Kyushu district of Japan between 2005 and 2006 were measured, and 100 strains for which carbapenem MICs were < or =0.5-32 microg/ml were selected. In this study, MIC was measured by two methods, i.e., the common serial twofold dilution method and an integrated concentration method, in which the concentration was changed, in increments of 2 microg/ml, from 2 to 16 microg/ml. The MIC(50)/MIC(90) values for imipenem, meropenem, biapenem, doripenem, and panipenem, respectively, with the former method were 8/16, 4/16, 4/16, 2/8, and 16/16 microg/ml; and the values were 6/10, 4/12, 4/10, 2/6, and 10/16 microg/ml with the latter method. The MIC data obtained with both methods were subjected to pharmacokinetic/pharmacodynamic (PK/PD) analysis with Monte Carlo simulation to calculate the probability of achieving the target of time above MIC (T>MIC) with each carbapenem. The probability of achieving 25% time above the MIC (T>MIC; % of T>MIC for dosing intervals) and 40% T>MIC against P. aeruginosa with any dosage regimen was higher with doripenem than with any other carbapenem tested. When the two sets of MIC data were subjected to PK/PD analysis, the difference between the two methods in the probability of achieving each % T>MIC was small, thus endorsing the validity of the serial twofold dilution method. PMID:18574662

  19. Pulmonary delivery of tobramycin-loaded nanostructured lipid carriers for Pseudomonas aeruginosa infections associated with cystic fibrosis.

    PubMed

    Moreno-Sastre, María; Pastor, Marta; Esquisabel, Amaia; Sans, Eulàlia; Viñas, Miguel; Fleischer, Aarne; Palomino, Esther; Bachiller, Daniel; Pedraz, José Luis

    2016-02-10

    Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies. PMID:26705155

  20. 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

  1. VAPOR PRESSURES, LIQUID MOLAR VOLUMES, VAPOR NON- IDEALITIES, AND CRITICAL PROPERTIES OF SOME FLUORINATED ETHERS: CF3OCF2OCF3, CF3OCF2 CF2H, c-CF2CF2CF2O, CF3OCF2H, AND CF3OCH3; AND OF CCl3F AND CF2ClH

    EPA Science Inventory

    Vapor pressures, compressibilities, expansivities, and molar volumes of the liquid phase have been measured between room temperature and the critical temperature for a series of fluorinated ethers: CF3OCF2OCF3, CF3OCF2CF2H, c-CF2CF2CF2O, CF3OCF2H, and CF3OCH3. Vapor-phase non-ide...

  2. 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

  3. Metallo-β-lactamase expression confers an advantage to Pseudomonas aeruginosa isolates compared with other β-lactam resistance mechanisms, favoring the prevalence of metallo-β-lactamase producers in a clinical environment.

    PubMed

    Corich, Lucia; Dolzani, Lucilla; Tonin, Enrico A; Vitali, Luca A; Lagatolla, Cristina

    2010-09-01

    The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings. PMID:20735174

  4. 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

  5. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  6. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections

    PubMed Central

    Winstanley, Craig; O’Brien, Siobhan; Brockhurst, Michael A.

    2016-01-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  7. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections.

    PubMed

    Winstanley, Craig; O'Brien, Siobhan; Brockhurst, Michael A

    2016-05-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  8. Pseudomonas aeruginosa Acquisition in Cystic Fibrosis Patients in Context of Otorhinolaryngological Surgery or Dentist Attendance: Case Series and Discussion of Preventive Concepts.

    PubMed

    Mainz, Jochen G; Gerber, Andrea; Lorenz, Michael; Michl, Ruth; Hentschel, Julia; Nader, Anika; Beck, James F; Pletz, Mathias W; Mueller, Andreas H

    2015-01-01

    Introduction. P. aeruginosa is the primary cause for pulmonary destruction and premature death in cystic fibrosis (CF). Therefore, prevention of airway colonization with the pathogen, ubiquitously present in water, is essential. Infection of CF patients with P. aeruginosa after dentist treatment was proven and dental unit waterlines were identified as source, suggesting prophylactic measures. For their almost regular sinonasal involvement, CF patients often require otorhinolaryngological (ORL) attendance. Despite some fields around ORL-procedures with comparable risk for acquisition of P. aeruginosa, such CF cases have not yet been reported. We present four CF patients, who primarily acquired P. aeruginosa around ORL surgery, and one around dentist treatment. Additionally, we discuss risks and preventive strategies for CF patients undergoing ORL-treatment. Perils include contact to pathogen-carriers in waiting rooms, instrumentation, suction, drilling, and flushing fluid, when droplets containing pathogens can be nebulized. Postsurgery mucosal damage and debridement impair sinonasal mucociliary clearance, facilitating pathogen proliferation and infestation. Therefore, sinonasal surgery and dentist treatment of CF patients without chronic P. aeruginosa colonization must be linked to repeated microbiological assessment. Further studies must elaborate whether all CF patients undergoing ORL-surgery require antipseudomonal prophylaxis, including nasal lavages containing antibiotics. Altogether, this underestimated risk requires structured prevention protocols. PMID:25866686

  9. Pseudomonas aeruginosa Acquisition in Cystic Fibrosis Patients in Context of Otorhinolaryngological Surgery or Dentist Attendance: Case Series and Discussion of Preventive Concepts

    PubMed Central

    Mainz, Jochen G.; Gerber, Andrea; Lorenz, Michael; Michl, Ruth; Hentschel, Julia; Nader, Anika; Beck, James F.; Pletz, Mathias W.; Mueller, Andreas H.

    2015-01-01

    Introduction. P. aeruginosa is the primary cause for pulmonary destruction and premature death in cystic fibrosis (CF). Therefore, prevention of airway colonization with the pathogen, ubiquitously present in water, is essential. Infection of CF patients with P. aeruginosa after dentist treatment was proven and dental unit waterlines were identified as source, suggesting prophylactic measures. For their almost regular sinonasal involvement, CF patients often require otorhinolaryngological (ORL) attendance. Despite some fields around ORL-procedures with comparable risk for acquisition of P. aeruginosa, such CF cases have not yet been reported. We present four CF patients, who primarily acquired P. aeruginosa around ORL surgery, and one around dentist treatment. Additionally, we discuss risks and preventive strategies for CF patients undergoing ORL-treatment. Perils include contact to pathogen-carriers in waiting rooms, instrumentation, suction, drilling, and flushing fluid, when droplets containing pathogens can be nebulized. Postsurgery mucosal damage and debridement impair sinonasal mucociliary clearance, facilitating pathogen proliferation and infestation. Therefore, sinonasal surgery and dentist treatment of CF patients without chronic P. aeruginosa colonization must be linked to repeated microbiological assessment. Further studies must elaborate whether all CF patients undergoing ORL-surgery require antipseudomonal prophylaxis, including nasal lavages containing antibiotics. Altogether, this underestimated risk requires structured prevention protocols. PMID:25866686

  10. Mitochondrial Ca2+-dependent NLRP3 activation exacerbates the Pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis.

    PubMed

    Rimessi, Alessandro; Bezzerri, Valentino; Patergnani, Simone; Marchi, Saverio; Cabrini, Giulio; Pinton, Paolo

    2015-01-01

    The common pathological manifestation of cystic fibrosis (CF) is associated with an excessive lung inflammatory response characterized by interleukin-1β accumulation. CF airway epithelial cells show an exacerbated pro-inflammatory response to Pseudomonas aeruginosa; however, it is unclear whether this heightened inflammatory response is intrinsic to cells lacking CF transmembrane conductance regulator (CFTR). Here we demonstrate that the degree and quality of the inflammatory response in CF are supported by P. aeruginosa-dependent mitochondrial perturbation, in which flagellin is the inducer and mitochondrial Ca(2+) uniporter (MCU) is a signal-integrating organelle member for NLRP3 activation and IL-1β and IL-18 processing. Our work elucidates the regulation of the NLRP3 inflammasome by mitochondrial Ca(2+) in the P. aeruginosa-dependent inflammatory response and deepens our understanding of the significance of mitochondria in the Ca(2+)-dependent control of inflammation. PMID:25648527

  11. Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans.

    PubMed

    Costello, Anne; Reen, F Jerry; O'Gara, Fergal; Callaghan, Máire; McClean, Siobhán

    2014-07-01

    Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens. PMID:24790091

  12. Pseudomonas aeruginosa in Cystic Fibrosis Patients With G551D-CFTR Treated With Ivacaftor

    PubMed Central

    Heltshe, Sonya L.; Mayer-Hamblett, Nicole; Burns, Jane L.; Khan, Umer; Baines, Arthur; Ramsey, Bonnie W.; Rowe, Steven M.

    2015-01-01

    Background. Ivacaftor improves outcomes in cystic fibrosis (CF) patients with the G551D mutation; however, effects on respiratory microbiology are largely unknown. This study examines changes in CF respiratory pathogens with ivacaftor and correlates them with baseline characteristics and clinical response. Methods. The G551D Observational Study enrolled a longitudinal observational cohort of US patients with CF aged 6 years and older with at least 1 copy of the G551D mutation. Results were linked with retrospective and prospective culture data in the US Cystic Fibrosis Foundation's National Patient Registry. Pseudomonas aeruginosa infection category in the year before and year after ivacaftor was compared and correlated with clinical findings. Results. Among 151 participants prescribed ivacaftor, 29% (26/89) who were culture positive for P. aeruginosa the year prior to ivacaftor use were culture negative the year following treatment; 88% (52/59) of those P. aeruginosa free remained uninfected. The odds of P. aeruginosa positivity in the year after ivacaftor compared with the year prior were reduced by 35% (odds ratio [OR], 0.65; P < .001). Ivacaftor was also associated with reduced odds of mucoid P. aeruginosa (OR, 0.77; P = .013) and Aspergillus (OR, 0.47; P = .039), but not Staphylococcus aureus or other common CF pathogens. Patients with intermittent culture positivity and higher forced expiratory volume in 1 second (FEV1) were most likely to turn culture negative. Reduction in P. aeruginosa was not associated with change in FEV1, body mass index, or hospitalizations. Conclusions. Pseudomonas aeruginosa culture positivity was significantly reduced following ivacaftor treatment. Efficacious CFTR modulation may contribute to lower frequency of culture positivity for P. aeruginosa and other respiratory pathogens, particularly in patients with less established disease. PMID:25425629

  13. Lipoxin A4 prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells by Pseudomonas aeruginosa.

    PubMed

    Higgins, Gerard; Fustero Torre, Coral; Tyrrell, Jean; McNally, Paul; Harvey, Brian J; Urbach, Valerie

    2016-06-01

    The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa. PMID:27084849

  14. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    PubMed Central

    de la Fuente-Núñez, César; Mansour, Sarah C.; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B.M.; Elliott, Melissa; Reffuveille, Fany; Speert, David P.; Reckseidler-Zenteno, Shauna L.; Shen, Ya; Haapasalo, Markus; Hancock, Robert E.W.

    2014-01-01

    Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients. PMID:26221537

  15. Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung▿ †

    PubMed Central

    Starkey, Melissa; Hickman, Jason H.; Ma, Luyan; Zhang, Niu; De Long, Susan; Hinz, Aaron; Palacios, Sergio; Manoil, Colin; Kirisits, Mary Jo; Starner, Timothy D.; Wozniak, Daniel J.; Harwood, Caroline S.; Parsek, Matthew R.

    2009-01-01

    Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV

  16. Photoinitiated oxidative addition of CF3I to gold(I) and facile aryl-CF3 reductive elimination.

    PubMed

    Winston, Matthew S; Wolf, William J; Toste, F Dean

    2014-05-28

    Herein we report the mechanism of oxidative addition of CF3I to Au(I), and remarkably fast Caryl-CF3 bond reductive elimination from Au(III) cations. CF3I undergoes a fast, formal oxidative addition to R3PAuR' (R = Cy, R' = 3,5-F2-C6H4, 4-F-C6H4, C6H5, 4-Me-C6H4, 4-MeO-C6H4, Me; R = Ph, R' = 4-F-C6H4, 4-Me-C6H4). When R' = aryl, complexes of the type R3PAu(aryl)(CF3)I can be isolated and characterized. Mechanistic studies suggest that near-ultraviolet light (λmax = 313 nm) photoinitiates a radical chain reaction by exciting CF3I. Complexes supported by PPh3 undergo reversible phosphine dissociation at 110 °C to generate a three-coordinate intermediate that undergoes slow reductive elimination. These processes are quantitative and heavily favor Caryl-I reductive elimination over Caryl-CF3 reductive elimination. Silver-mediated halide abstraction from all complexes of the type R3PAu(aryl)(CF3)I results in quantitative formation of Ar-CF3 in less than 1 min at temperatures as low as -10 °C. PMID:24836526

  17. Analysis and Characterization of Staphylococcus aureus Small Colony Variants Isolated From Cystic Fibrosis Patients in Austria.

    PubMed

    Masoud-Landgraf, Lilian; Zarfel, Gernot; Kaschnigg, Tanja; Friedl, Simone; Feierl, Gebhard; Wagner-Eibel, Ute; Eber, Ernst; Grisold, Andrea J; Kittinger, Clemens

    2016-05-01

    Cystic fibrosis (CF) is the most common hereditary lung disease in the Caucasian population, characterized by viscous bronchial secretion, consecutive defective mucociliary clearance, and unavoidable colonization with microorganisms. Besides Pseudomonas aeruginosa, Staphylococcus aureus is the most common bacterial species colonizing the CF respiratory tract. Under antibiotic pressure S. aureus is able to switch to small colony variants (SCV). These small colony variants can invade epithelial cells, overcome antibiotic therapy inside the cells and can be the starting point for extracellular recolonization. The aim of the present study was the isolation and characterization of S. aureus small colony variants from Austrian cystic fibrosis patients. Samples collected from 147 patients were screened for the presence of S. aureus wild-type and small colony variants. Antibiotic susceptibility testing and determination of the small colony variants causing auxotrophism were performed. Wild-type isolates were assigned to corresponding small colony variants with spa typing. In total, 17 different small colony variant isolates and 12 corresponding wild-type isolates were obtained. 13 isolates were determined thymidine auxotroph, 2 isolates were auxotroph for hemin, and none of the tested isolates was auxotroph for both, respectively. The presence of SCVs is directly related to a poor clinical outcome, therefore a monitoring of SCV prevalence is recommended. This study revealed rather low SCV ratios in CF patients compared to other countries. PMID:26821237

  18. Initial Pseudomonas aeruginosa Treatment Failure is Associated with Exacerbations in Cystic Fibrosis

    PubMed Central

    Mayer-Hamblett, Nicole; Kronmal, Richard A.; Gibson, Ronald L.; Rosenfeld, Margaret; Retsch-Bogart, George; Treggiari, Miriam M.; Burns, Jane L.; Khan, Umer; Ramsey, Bonnie W

    2011-01-01

    Rationale The risk of pulmonary exacerbation following Pseudomonas aeruginosa (Pa) acquisition in children with cystic fibrosis (CF) is unknown. Objectives To determine if failure of antibiotic therapy to eradicate Pa and frequency of Pa recurrence are associated with increased exacerbation risk. Methods The cohort included 282 children with CF who participated in the EPIC trial ages 1–12 with newly acquired Pa, defined as either a first lifetime Pa positive respiratory culture or positive after two years of negative cultures (past isolation of Pa but >2 years prior to the trial). All received antibiotics to promote initial eradication followed by 15 months of intermittent maintenance antibiotics. Quarterly cultures were used to define initial eradication success and subsequent number of Pa recurrences. A standardized symptom-based definition of exacerbation was utilized. Cox proportional hazards models were used to estimate exacerbation risk. Results Failure to initially eradicate Pa was associated with exacerbation risk (hazard ratio [HR]: 2.49, 95% confidence interval [CI] 1.26,4.93). In 245/282 with successful initial eradication during the trial, past isolation of Pa >2 years before the trial was the most significant predictor of exacerbation (HR 1.62, 95% CI 1.12,2.35). In 37/282 who failed initial eradication, persistent Pa during the maintenance phase (1 or more Pa recurrences after failure to initially eradicate) added even greater exacerbation risk (HR 4.13, 95% CI 1.28, 13.32). Conclusions Children with CF who fail to eradicate after initial antibiotic treatment are at higher risk of subsequent exacerbation, suggesting clinical benefit to successful early eradication of Pa infection. PMID:21830317

  19. In Vivo Efficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa.

    PubMed

    Pawar, Vinay; Komor, Uliana; Kasnitz, Nadine; Bielecki, Piotr; Pils, Marina C; Gocht, Benjamin; Moter, Annette; Rohde, Manfred; Weiss, Siegfried; Häussler, Susanne

    2015-08-01

    Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds. PMID:26055372

  20. Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa.

    PubMed

    Smania, Andrea M; Segura, Ignacio; Pezza, Roberto J; Becerra, Cecilia; Albesa, Inés; Argaraña, Carlos E

    2004-05-01

    S diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed. PMID:15133095

  1. The MerR-Like Regulator BrlR Impairs Pseudomonas aeruginosa Biofilm Tolerance to Colistin by Repressing PhoPQ

    PubMed Central

    Chambers, Jacob R.

    2013-01-01

    While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance. PMID:23935054

  2. 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

  3. CF.sub.4 laser

    DOEpatents

    Wittig, Curt; Tiee, Joe J.

    1979-01-01

    A CF.sub.4 laser for producing near 16 .mu.m radiation utilizing a line tunable CO.sub.2 laser as an optical pumping source. The device uses a cryogenically cooled optically pumped cell containing molecular CF.sub.4 gas. An optical resonant cavity formed around the optically pumped cell induces oscillations of near 16 .mu.m radiation from the .nu..sub.2 +.nu..sub.4 .fwdarw..nu..sub.2 transition in the molecular CF.sub.4 gas.

  4. [Approach to directed therapy after knowledge of the isolate: carbapenemase-producing Enterobacteriaceae, multidrug-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii].

    PubMed

    Martínez, J A

    2016-09-01

    Directed treatment of infections due to multidrug-resistant Gram-negative bacilli is a difficult task, since it requires the use of a limited number of antibiotics that are often more toxic and possibly less efficacious than β-lactams and fluoroquinolones. Furthermore, there are very few controlled trials informing on the relative efficacy of different therapeutic strategies. As a general rule, it is recommended to use at least two active drugs or a combination with proven synergistic activity in vitro, because several observational studies have associated this practice with better outcomes and as a measure to potentially curb the emergence of further resistance. It is already available a new cephalosporin active against most strains of Pseudomonas aeruginosa resistant to ceftazidime due to derepression of ampC and in the near future an effective inhibitor of class A, class C and OXA-48 will be available which combined with ceftazidime is expected to mean a significant addition to the armamentarium against Gram-negative bacilli with these resistance determinants. PMID:27608310

  5. A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry

    PubMed Central

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H.; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S.; Schellenberger, Wolfgang; Rodloff, Arne C.; Eschrich, Klaus

    2012-01-01

    Summary Background Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. Material/Methods We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. Results Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. Conclusions This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics. PMID:22936198

  6. Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation.

    PubMed

    Price, Katherine E; Orazi, Giulia; Ruoff, Kathryn L; Hebert, Wesley P; O'Toole, George A; Mastoridis, Paul

    2015-01-01

    Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting. PMID:26506004

  7. 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.

  8. 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

  9. Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

    PubMed Central

    Price, Katherine E.; Naimie, Amanda A.; Griffin, Edward F.; Bay, Charles

    2015-01-01

    ABSTRACT Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways. IMPORTANCE Deep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions

  10. Ambroxol inhibits mucoid conversion of Pseudomonas aeruginosa and contributes to the bactericidal activity of ciprofloxacin against mucoid P. aeruginosa biofilms.

    PubMed

    Wang, Wenlei; Yu, Jialin; He, Yu; Wang, Zhengli; Li, Fang

    2016-07-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that can cause severe infections in immunocompromised individuals. Because it forms biofilms, which protect against host immune attack and increase resistance to conventional antibiotics, mucoid P. aeruginosa is nearly impossible to eradicate. Moreover, mucoid conversion of P. aeruginosa in cystic fibrosis (CF) patients leads to poor outcomes. This conversion is mainly due to mucA gene mutation, which is thought to be induced by polymorphonuclear leukocytes (PMNs) and the reactive oxygen species they release. Ambroxol, a mucolytic agent with antioxidant characteristics, is used clinically, and this compound has recently been demonstrated to possess anti-biofilm properties. In this study, we found that ambroxol inhibits the H2 O2 -mediated conversion of P. aeruginosa from a non-mucoid to a mucoid phenotype, an effect that is due to its antioxidant property against H2 O2 . Furthermore, the bactericidal activity of ciprofloxacin against mucoid P. aeruginosa biofilms was increased in vitro when used in combination with ambroxol. PMID:27102839

  11. Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates.

    PubMed

    Gallagher, Lauren C; Roundtree, Sylvester S; Lancaster, Diana P; Rudin, Susan D; Bard, Jennifer Dien; Roberts, Amity L; Marshall, Steven H; Bonomo, Robert A; Sullivan, Kaede V

    2015-10-01

    This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with blaNDM, blaIMP, and blaOXA-48. PMID:26269624

  12. 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

  13. Pseudomonas aeruginosa colonization in patients with spinal cord injuries.

    PubMed Central

    Gilmore, D S; Bruce, S K; Jimenez, E M; Schick, D G; Morrow, J W; Montgomerie, J Z

    1982-01-01

    The prevalence of Pseudomonas aeruginosa colonization of patients with spinal cord injury was studied annually from 1976 to 1980. The urethra, perineum, rectum, drainage bag, and urine of patients on the spinal cord injury service were cultured. A total of 224 men and 32 women were studied. Most patients were managed with an external urinary collection system or padding, with or without intermittent catheterization. P. aeruginosa was cultured from one or more body sites (urethra, perineum, or rectum) in 65% of men and 18% of women. Drainage bags on the beds were frequently colonized with P. aeruginosa (73%). Significant bacteriuria with P. aeruginosa was present in 19% of the men and 13% of the women. P. aeruginosa colonization of body sites in men was closely associated with the use of an external urinary collection system. Significantly greater urethral and perineal colonization was found in men using an external urinary collection system. P. aeruginosa serotype 11 was the predominant serotype for the first 3 years, and the number of patients colonized with serotype 11 increased with length of hospital stay. The prevalence of serotype 11 significantly decreased in the last 2 years. The antibiotic susceptibility of the strains of P. aeruginosa isolated from these patients did not change in the 5 years, except that there was increasing susceptibility to carbenicillin in later years. This increasing susceptibility to carbenicillin was a reflection of a decreased prevalence of serotype 11 in these patients, since serotype 11 was more resistant than other serotypes to carbenicillin. PMID:6818251

  14. 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

  15. Pseudomonas Aeruginosa: Resistance to the Max

    PubMed Central

    Poole, Keith

    2011-01-01

    Pseudomonas aeruginosa is intrinsically resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism. Resistance to multiple classes of antimicrobials (multidrug resistance) in particular is increasingly common in P. aeruginosa, with a number of reports of pan-resistant isolates treatable with a single agent, colistin. Acquired resistance in this organism is multifactorial and attributable to chromosomal mutations and the acquisition of resistance genes via horizontal gene transfer. Mutational changes impacting resistance include upregulation of multidrug efflux systems to promote antimicrobial expulsion, derepression of ampC, AmpC alterations that expand the enzyme's substrate specificity (i.e., extended-spectrum AmpC), alterations to outer membrane permeability to limit antimicrobial entry and alterations to antimicrobial targets. Acquired mechanisms contributing to resistance in P. aeruginosa include β-lactamases, notably the extended-spectrum β-lactamases and the carbapenemases that hydrolyze most β-lactams, aminoglycoside-modifying enzymes, and 16S rRNA methylases that provide high-level pan-aminoglycoside resistance. The organism's propensity to grow in vivo as antimicrobial-tolerant biofilms and the occurrence of hypermutator strains that yield antimicrobial resistant mutants at higher frequency also compromise anti-pseudomonal chemotherapy. With limited therapeutic options and increasing resistance will the untreatable P. aeruginosa infection soon be upon us? PMID:21747788

  16. More than 10 years' continuous oral treatment with specific immunoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report.

    PubMed

    Nilsson, Elin; Kollberg, Hans; Johannesson, Marie; Wejåker, Per-Erik; Carlander, David; Larsson, Anders

    2007-06-01

    Immunotherapy with specific antibodies is an alternative to antibiotics for the prevention of infections in humans and animals. We have used orally administered immunoglobulin Y (IgY) preparations, purified from eggs of hens immunized with Pseudomonas aeruginosa bacteria, to prevent pulmonary P. aeruginosa infections in a group of patients with cystic fibrosis (CF). Respiratory infections are major problems for CF patients because of the thick mucus in the airways, and chronic P. aeruginosa lung infections occur in virtually all CF patients and cause morbidity and mortality. The IgY-treated group had only 2.5 P. aeruginosa-positive sputum cultures per 100 months, and none of the IgY-treated patients became chronically colonized with P. aeruginosa. In the control group, 13.7 of the cultures per 100 months were positive for P. aeruginosa, and 24% of patients became chronically colonized with P. aeruginosa. The first enrolled patient in this study has now been treated continuously for more than 10 years. During the first 8 years she only had four P. aeruginosa-positive cultures. After 8 years she became chronically infected, but still after 10 years the bacteria have not turned mucoid. No negative side effects of IgY treatment have been noted during these 10 years. To our knowledge this is the longest treatment with specific yolk antibodies for therapeutic purposes. PMID:17651078

  17. Cyanide in bronchoalveolar lavage is not diagnostic for Pseudomonas aeruginosa in children with cystic fibrosis.

    PubMed

    Stutz, M D; Gangell, C L; Berry, L J; Garratt, L W; Sheil, B; Sly, P D

    2011-03-01

    Early detection of the cyanobacterium Pseudomonas aeruginosa in the lungs of young children with cystic fibrosis (CF) is considered the key to delaying chronic pulmonary disease. We investigated whether cyanide in bronchoalveolar lavage (BAL) fluid could be used as an early diagnostic biomarker of infection. Cyanide was measured in 226 BAL samples (36 P. aeruginosa infected) obtained from 96 infants and young children with CF participating in an early surveillance programme involving annual BAL. Cyanide was detected in 97.2% of P. aeruginosa infected and 60.5% of uninfected samples. Cyanide concentrations were significantly higher in BALs infected with P. aeruginosa (median (25th-75th percentile) 27.3 (22.1-33.3) μM) than those which were not (17.2 (7.85-23.0) μM, p<0.001). The best sensitivity, specificity, positive and negative predictive values were obtained with a cut-off concentration of 20.6 μM, and were 83%, 66%, 32% and 96%, respectively. Neutrophil number in BAL was a significant predictor of cyanide concentration (p<0.001). Cyanide concentration can distinguish between P. aeruginosa infected and uninfected BALs as a group, but not individually; therefore, cyanide is a poor diagnostic biomarker of P. aeruginosa infection. Cyanide levels in BAL are related to the level of neutrophilic inflammation. PMID:20562125

  18. Characterisation of Pseudomonas aeruginosa related to bovine mastitis.

    PubMed

    Park, Hye Rim; Hong, Min Ki; Hwang, Sun Young; Park, Young Kyung; Kwon, Ka Hee; Yoon, Jang Won; Shin, Sook; Kim, Jae Hong; Park, Yong Ho

    2014-03-01

    Pseudomonas aeruginosa is one of the causative pathogens of bovine mastitis. Most P. aeruginosa strains possess the type III secretion system (TTSS), which may increase somatic cell counts (SCCs) in milk from mastitis-affected cows. Moreover, most of P. aeruginosa cells can form biofilms, thereby reducing antibiotic efficacy. In this study, the presence and effect of TTSS-related genotypes on increase of SCCs among 122 P. aeruginosa isolates obtained from raw milk samples from mastitis-affected cows and their antibiotic susceptibility at planktonic and biofilm status were investigated. Based on the presence of TTSS-related genes a total of 82.7% of the isolates were found to harbour exoU and/or exoS genes, including the invasive (exoU-/exoS+, 69.4%), cytotoxic (exoU+/exoS-, 8.3%) and cytotoxic/invasive strains (exoU+/ exoS+, 5.0%). Milk containing exoS-positive isolates had higher SCCs than those containing exoS-negative isolates. The majority of isolates showed gentamicin, amikacin, meropenem and ciprofloxacin susceptibility at planktonic status. However, the susceptibility was decreased at the biofilm status. Based on minimum biofilm eradication concentration (MBEC)/minimum inhibitory concentration (MIC) ratios, the range of change in antibiotic susceptibility varied widely depending on the antibiotics (from ≥ 3.1-fold to ≥ 475.0-fold). In conclusion, most P. aeruginosa isolates studied here had a genotype related to increase in SCCs. The efficiency of antibiotic therapy against P. aeruginosa-related bovine mastitis could be improved by analysing both the MBEC and the MIC of isolates. PMID:24334080

  19. GBA2-Encoded β-Glucosidase Activity Is Involved in the Inflammatory Response to Pseudomonas aeruginosa

    PubMed Central

    Lampronti, Ilaria; Marchetti, Nicola; Aureli, Massimo; Bassi, Rosaria; Giri, Maria Grazia; Bezzerri, Valentino; Lovato, Valentina; Cantù, Cinzia; Munari, Silvia; Cheng, Seng H.; Cavazzini, Alberto; Gambari, Roberto; Sonnino, Sandro; Cabrini, Giulio; Dechecchi, Maria Cristina

    2014-01-01

    Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2. PMID:25141135

  20. Adsorption of CF4 on graphite preplated with a monolayer of CF3Cl

    NASA Astrophysics Data System (ADS)

    Thomas, Petros; Velazquez, Daniel; Hess, George B.

    2011-03-01

    We report a study of the adsorption of CF4 on graphite preplated with a monolayer of CF3Cl, using infrared reflection absorption spectroscopy combined with ellipsometry. The saturated vapor pressure of CF3Cl is nearly 3 orders of magnitude smaller than that of CF4 at the same temperature, so the main control variables are the temperature and the pressure (or chemical potential) of CF4, together with the initial coverage of CF3Cl. The temperature range covered is 60-105 K. We find that, if the initial monolayer of CF3Cl is liquid, CF4 continuously displaces CF3Cl by substitution in the monolayer. If the initial monolayer of CF3Cl is solid, due to either lower temperature or compression, CF4 condenses as a second layer on the top of the CF3Cl layer, with only slight mixing with the original layer. This behavior persists to multiple layers of CF4.

  1. Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

    SciTech Connect

    Keravec, Marlène; Mounier, Jérôme; Prestat, Emmanuel; Vallet, Sophie; Jansson, Janet K.; Burgaud, Gaëtan; Rosec, Sylvain; Gouriou, Stéphanie; Rault, Gilles; Coton, Emmanuel; Barbier, Georges; Héry-Arnaud, Geneviève

    2015-08-09

    Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly more prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.

  2. 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

  3. Effect of tannin extract against Pseudomonas aeruginosa producing metallo beta-lactamase.

    PubMed

    Ghafourian, S; Mohebi, R; Sekawi, Z; Raftari, M; Neela, V; Ghafourian, E; Aboualigalehdari, E; Rahbar, M; Sadeghifard, N

    2012-01-01

    Carbapenems are the most potent beta-lactam agents with a broad-spectrum activity against Gram-negative and Gram-positive bacteria. They are stable in the presence of penicillinases and cephalosporinases. This study was focused on frequency of metallo beta- lactamase (MBL) among Pesudomonas aeruginosa strains isolated in patients with urinary tract infection, effect of tannin against PA positive strains which produced blaVIM or blaIMP and both of these genes (Species). Detection of MBL was performed by phonotypic and genotypic methods. Tannin extract was tested against P. aeruginosa producing MBL. During the study period, 240 P. aeruginosa isolates were identified. Among them 64 (26.6 percent) isolates were imipenem non-susceptible and confirmed by imipenem/EDTA. Our results revealed that the growth of blaVIM positive P. aeruginosa inhibited at 15 microg/ml concentration. The experiment repeated for blaIMP-positive P. aeruginosa and P. aeruginosa which harbored blaIMP and blaVIM, the results showed 35 microg/ml was the best concentration for inhibition of P. aeruginosa-positive blaIMP and also P. aeruginosa blaIMP and blaVIM. In conclusion, tannin was effective against P. aeruginosa producing blaVIM and blaIMP and both of them so it can be substituted with common antibiotics. The result showed significantly P. aeruginosa-harbored blaIMP was more responsible for imipenem resistance than P. aeruginosa-positive blaVIM. Interestingly, tannin was more effective against MBL-P. aeruginosa in comparison with current antibiotics. PMID:22824750

  4. Normal and Cystic Fibrosis Human Bronchial Epithelial Cells Infected with Pseudomonas aeruginosa Exhibit Distinct Gene Activation Patterns

    PubMed Central

    Balloy, Viviane; Varet, Hugo; Dillies, Marie-Agnès; Proux, Caroline; Jagla, Bernd; Coppée, Jean-Yves; Tabary, Olivier; Corvol, Harriet; Chignard, Michel; Guillot, Loïc

    2015-01-01

    Background and Aims In cystic fibrosis (CF), Pseudomonas aeruginosa is not eradicated from the lower respiratory tract and is associated with epithelial inflammation that eventually causes tissue damage. To identify the molecular determinants of an effective response to P. aeruginosa infection, we performed a transcriptomic analysis of primary human bronchial epithelial cells from healthy donors (CTRL) 2, 4, and 6 h after induced P. aeruginosa infection. Compared to noninfected cells, infected cells showed changes in gene activity, which were most marked 6 h postinfection and usually consisted in upregulation. Results By comparing for each time point of infection, the transcriptomic response of epithelial cells from CF patients and healthy donors, we identified 851, 638, 667, and 980 differentially expressed genes 0, 2, 4, and 6 h postinfection, respectively. Gene selection followed by bioinformatic analysis showed that most of the differentially expressed genes, either up- or downregulated, were in the protein-binding and catalytic gene-ontology categories. Finally, we established that the protein products of the genes exhibiting the greatest differential upregulation (CSF2, CCL2, TNF, CSF3, MMP1, and MMP10) between CF patients and CTRL were produced in higher amounts by infected cells from CF patients versus CTRL. Conclusions The differentially expressed genes in CF patients may constitute a signature for a detrimental inflammatory response and for an inefficient P. aeruginosa host-cell response. PMID:26485688

  5. The MerR-Like Transcriptional Regulator BrlR Contributes to Pseudomonas aeruginosa Biofilm Tolerance

    PubMed Central

    Liao, Julie

    2012-01-01

    Biofilms are composed of surface-attached microbial communities. A hallmark of biofilms is their profound tolerance of antimicrobial agents. While biofilm drug tolerance has been considered to be multifactorial, our findings indicate, instead, that bacteria within biofilms employ a classical regulatory mechanism to resist the action of antimicrobial agents. Here we report that the transcriptional regulator BrlR, a member of the MerR family of multidrug transport activators, plays a role in the high-level drug tolerance of biofilms formed by Pseudomonas aeruginosa. Expression of brlR was found to be biofilm specific, with brlR inactivation not affecting biofilm formation, motility, or pslA expression but increasing ndvB expression. Inactivation of brlR rendered biofilms but not planktonic cells grown to exponential or stationary phase significantly more susceptible to hydrogen peroxide and five different classes of antibiotics by affecting the MICs and the recalcitrance of biofilms to killing by microbicidal antimicrobial agents. In contrast, overexpression of brlR rendered both biofilms and planktonic cells more tolerant to the same compounds. brlR expression in three cystic fibrosis (CF) isolates was elevated regardless of the mode of growth, suggesting a selection for constitutive brlR expression upon in vivo biofilm formation associated with chronic infections. Despite increased brlR expression, however, isolate CF1-8 was as susceptible to tobramycin as was a ΔbrlR mutant because of a nonsense mutation in brlR. Our results indicate for the first time that biofilms employ a specific regulatory mechanism to resist the action of antimicrobial agents in a BrlR-dependent manner which affects MIC and recalcitrance to killing by microbicidal antimicrobial agents. PMID:22730129

  6. 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

  7. MOLECULAR CHARACTERIZATION OF 'PSEUDOMONAS AERUGINOSA' BACTERIOPHAGES: IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL VIRUS B86

    EPA Science Inventory

    The authors have characterized a new phage, B86, of Pseudomonas aeruginosa isolated from nature. It is a temperate, uv-inducible, generalized transducing phage. To determine the relatedness of his phage to other characterized P. aeruginosa phages, DNA homology studies were carrie...

  8. Complete Genome Sequences of Broad-Host-Range Pseudomonas aeruginosa Bacteriophages ΦR18 and ΦS12-1

    PubMed Central

    Furusawa, Takaaki; Higuchi, Hidetoshi; Usui, Masaru; Maruyama, Fumito; Nakagawa, Ichiro; Yokota, Hiroshi; Tamura, Yutaka

    2016-01-01

    Pseudomonas aeruginosa is an important cause of racehorse keratitis. Bacteriophage therapy has the potential to aid in the prevention and treatment of diseases caused by P. aeruginosa. We present here the complete genome sequences of two phages, ΦR18 and ΦS12-1, which exhibit infectivity for a broad range of P. aeruginosa isolates. PMID:27151780

  9. Continued transmission of Pseudomonas aeruginosa from a wash hand basin tap in a critical care unit.

    PubMed

    Garvey, M I; Bradley, C W; Tracey, J; Oppenheim, B

    2016-09-01

    Pseudomonas aeruginosa is an important nosocomial pathogen, colonizing hospital water supplies including taps and sinks. We report a cluster of P. aeruginosa acquisitions during a period of five months from tap water to patients occupying the same burns single room in a critical care unit. Pseudomonas aeruginosa cultured from clinical isolates from four different patients was indistinguishable from water strains by pulsed-field gel electrophoresis. Water outlets in critical care may be a source of P. aeruginosa despite following the national guidance, and updated guidance and improved control measures are needed to reduce the risks of transmission to patients. PMID:27249962

  10. YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

    PubMed Central

    Malone, Jacob G.; Jaeger, Tina; Spangler, Christian; Ritz, Daniel; Spang, Anne; Arrieumerlou, Cécile; Kaever, Volkhard; Landmann, Regine; Jenal, Urs

    2010-01-01

    During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important

  11. The Effect of Strict Segregation on Pseudomonas aeruginosa in Cystic Fibrosis Patients

    PubMed Central

    van Mansfeld, Rosa; de Vrankrijker, Angelica; Brimicombe, Roland; Heijerman, Harry; Teding van Berkhout, Ferdinand; Spitoni, Cristian; Grave, Sanne; van der Ent, Cornelis; Wolfs, Tom; Willems, Rob; Bonten, Marc

    2016-01-01

    Introduction Segregation of patients with cystic fibrosis (CF) was implemented to prevent chronic infection with epidemic Pseudomonas aeruginosa strains with presumed detrimental clinical effects, but its effectiveness has not been carefully evaluated. Methods The effect of strict segregation on the incidence of P. aeruginosa infection in CF patients was investigated through longitudinal protocolized follow-up of respiratory tract infection before and after segregation. In two nested cross-sectional studies in 2007 and 2011 the P. aeruginosa population structure was investigated and clinical parameters were determined in patients with and without infection with the Dutch epidemic P. aeruginosa clone (ST406). Results Of 784 included patients 315 and 382 were at risk for acquiring chronic P. aeruginosa infection before and after segregation. Acquisition rates were, respectively, 0.14 and 0.05 per 1,000 days at risk (HR: 0.66, 95% CI [0.2548–1.541]; p = 0.28). An exploratory subgroup analysis indicated lower acquisition after segregation in children < 15 years of age (HR: 0.43, 95% CI[0.21–0.95]; p = 0.04). P. aeruginosa population structure did not change after segregation and ST406 was not associated with lung function decline, death or lung transplantation. Conclusions Strict segregation was not associated with a statistically significant lower acquisition of chronic P. aeruginosa infection and ST406 was not associated with adverse clinical outcome. After segregation there were no new acquisitions of ST406. In an unplanned exploratory analysis chronic acquisition of P. aeruginosa was lower after implementation of segregation in patients under 15 years of age. PMID:27280467

  12. Transferable imipenem resistance in Pseudomonas aeruginosa.

    PubMed Central

    Watanabe, M; Iyobe, S; Inoue, M; Mitsuhashi, S

    1991-01-01

    We isolated an imipenem-resistant strain, GN17203, of Pseudomonas aeruginosa. The strain produced a beta-lactamase that hydrolyzed imipenem. The beta-lactamase was encoded by a 31-MDa plasmid, pMS350, which belongs to incompatibility group P-9. The plasmic conferred resistance to beta-lactams, gentamicin, and sulfonamide and was transferable by conjugation to P. aeruginosa but not to Escherichia coli. The molecular weight of the purified enzyme was estimated to be 28,000, and the isoelectric point was 9.0. The enzyme showed a broad substrate profile, hydrolyzing imipenem, oxyiminocephalosporins, 7-methoxycephalosporins, and penicillins. The enzyme activity was inhibited by EDTA, iodine, p-chloromercuribenzoate, CuSO4, and HgCl2 but not by clavulanic acid or sulbactam. Images PMID:1901695

  13. Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition

    PubMed Central

    Peek, Mary E.; Bhatnagar, Abhinav; McCarty, Nael A.; Zughaier, Susu M.

    2012-01-01

    Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs. PMID:22973307

  14. Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition.

    PubMed

    Peek, Mary E; Bhatnagar, Abhinav; McCarty, Nael A; Zughaier, Susu M

    2012-01-01

    Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs. PMID:22973307

  15. Graphite moderated (252)Cf source.

    PubMed

    Sajo-Bohus, Laszlo; Barros, Haydn; Greaves, Eduardo D; Vega-Carrillo, Hector Rene

    2015-06-01

    The Thorium molten-salt reactor is an attractive and affordable nuclear power option for developing countries with insufficient infrastructure and limited technological capability. In the aim of personnel training and experience gathering at the Universidad Simon Bolivar there is in progress a project of developing a subcritical thorium liquid-fuel reactor. The neutron source to run this subcritical reactor is a (252)Cf source and the reactor will use high-purity graphite as moderator. Using the MCNP5 code the neutron spectra of the (252)Cf in the center of the graphite moderator has been estimated along the channel where the liquid thorium salt will be inserted; also the ambient dose equivalent due to the source has been determined around the moderator. PMID:25770393

  16. Nosocomial outbreak of OXA-18-producing Pseudomonas aeruginosa in Tunisia.

    PubMed

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

    2007-08-01

    Following systematic screening for ceftazidime-resistant (CAZ-R) Pseudomonas aeruginosa, 24 isolates producing extended-spectrum beta-lactamase (ESBL) were recovered during a 24-month period at the National Bone Marrow Transplant Centre of Tunisia. These isolates were from seven immunocompromised patients and from environmental swabs. ESBLs inhibited by clavulanic acid were detected by double-disk diffusion tests. Isoelectric focusing revealed that these isolates produced two to four beta-lactamases with pIs of 5.5, 6.1, 6.4, 7.6 or 8.2, and PCR detected the presence of bla(OXA-18), bla(SHV) and bla(TEM) genes in 24, 21 and two isolates, respectively. Pulsed-field gel electrophoresis defined two dominant genotypic groups: group A (16 isolates) and group B (four isolates). Sequencing of PCR products from representative isolates identified the bla(OXA-18) gene and revealed nucleotide sequences belonging to the bla(SHV-1) and bla(TEM-1) genes. Isolates producing OXA-18 belonged to genomic group A and were isolated from four immunocompromised patients in the haematology and graft units, and from two wash-basins in the graft unit. No immunocompromised patient harboured the clonal epidemic strain upon admission. This is the first report of the OXA-18-type ESBL in P. aeruginosa in Tunisia, and the first description of an outbreak caused by an OXA-18-producing strain of P. aeruginosa. PMID:17610599

  17. Monoclonal antibodies to Pseudomonas aeruginosa ferripyochelin-binding protein.

    PubMed Central

    Sokol, P A; Woods, D E

    1986-01-01

    Hybridomas secreting specific monoclonal antibodies against the Pseudomonas aeruginosa ferripyochelin-binding protein (FBP) were isolated. These monoclonal antibodies reacted with FBP in immunoblots of outer membrane preparations from all serotypes of P. aeruginosa. Two of the monoclonal antibodies also reacted with FBP in strains of P. putida, P. fluorescens, and P. stutzeri. These antibodies did not react with outer membranes of P. cepacia, "P. multivorans," P. maltophilia, or other gram-negative organisms. The monoclonal antibodies were opsonophagocytic and blocked the binding of [59Fe]ferripyochelin to isolated outer membranes of strain PAO. By indirect immunofluorescence techniques, the monoclonal antibodies were used to demonstrate that FBP is present on the cell surface of P. aeruginosa cells grown in low-iron but not high-iron medium. These observations were confirmed by using 125I in surface-labeling techniques. Images PMID:3091506

  18. Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa.

    PubMed

    Green, S K; Schroth, M N; Cho, J J; Kominos, S K; Vitanza-jack, V B

    1974-12-01

    Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture. PMID:4217591

  19. Agricultural Plants and Soil as a Reservoir for Pseudomonas aeruginosa

    PubMed Central

    Green, Sylvia K.; Schroth, Milton N.; Cho, John J.; Kominos, Spyros D.; Vitanza-Jack, Vilma B.

    1974-01-01

    Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture. PMID:4217591

  20. Vacuum-UV negative photoion spectroscopy of CF3Cl, CF3Br, and CF3I

    NASA Astrophysics Data System (ADS)

    Simpson, M. J.; Tuckett, R. P.; Dunn, K. F.; Hunniford, C. A.; Latimer, C. J.

    2009-05-01

    Using synchrotron radiation, negative ions are detected by mass spectrometry following vacuum-UV photoexcitation of trifluorochloromethane (CF3Cl), trifluorobromomethane (CF3Br), and trifluoroiodomethane (CF3I). The anions F-, X-, F2-, FX-, CF-, CF2-, and CF3- are observed from all three molecules, where X =Cl, Br, or I, and their ion yields recorded in the range of 8-35 eV. With the exception of Br- and I-, the anions observed show a linear dependence of signal with pressure, showing that they arise from unimolecular ion-pair dissociation. Dissociative electron attachment, following photoionization of CF3Br and CF3I as the source of low-energy electrons, is shown to dominate the observed Br- and I- signals, respectively. Cross sections for ion-pair formation are put onto an absolute scale by calibrating the signal strengths with those of F- from both SF6 and CF4. These anion cross sections are normalized to vacuum-UV absorption cross sections, where available, and the resulting quantum yields are reported. Anion appearance energies are used to calculate upper limits to 298 K bond dissociation energies for Do(CF3-X), which are consistent with literature values. We report new data for Do(CF2I+-F)≤2.7±0.2 eV and ΔfH298o(CF2I+)≤(598±22) kJ mol-1. No ion-pair formation is observed below the ionization energy of the parent molecule for CF3Cl and CF3Br, and only weak signals (in both I- and F-) are detected for CF3I. These observations suggest that neutral photodissociation is the dominant exit channel to Rydberg state photoexcitation at these lower energies.

  1. Selective Sweeps and Parallel Pathoadaptation Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung

    PubMed Central

    Diaz Caballero, Julio; Clark, Shawn T.; Coburn, Bryan; Zhang, Yu; Wang, Pauline W.; Donaldson, Sylva L.; Tullis, D. Elizabeth; Yau, Yvonne C. W.; Waters, Valerie J.; Hwang, David M.

    2015-01-01

    ABSTRACT Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage originated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with recurrent selective sweeps. While we cannot identify the specific mutations responsible for the origins of the clonal lineages, we find that the majority of mutations occur in loci previously associated with virulence and resistance. Additionally, approximately one-third of all mutations occur in loci that are mutated multiple times, highlighting the importance of parallel pathoadaptation. One such locus is the gene encoding penicillin-binding protein 3, which received three independent mutations. Our functional analysis of these alleles shows that they provide differential fitness benefits dependent on the antibiotic under selection. These data reveal that bacterial populations can undergo extensive and dramatic changes that are not revealed by lower-resolution analyses. PMID:26330513

  2. The Pseudomonas aeruginosa Transcriptional Landscape Is Shaped by Environmental Heterogeneity and Genetic Variation

    PubMed Central

    Schniederjans, Monika; Khaledi, Ariane; Hornischer, Klaus; Schulz, Sebastian; Bielecka, Agata; Eckweiler, Denitsa; Pohl, Sarah; Häussler, Susanne

    2015-01-01

    ABSTRACT Phenotypic variability among bacteria depends on gene expression in response to different environments, and it also reflects differences in genomic structure. In this study, we analyzed transcriptome sequencing (RNA-seq) profiles of 151 Pseudomonas aeruginosa clinical isolates under standard laboratory conditions and of one P. aeruginosa type strain under 14 different environmental conditions. Our approach allowed dissection of the impact of the genetic background versus environmental cues on P. aeruginosa gene expression profiles and revealed that phenotypic variation was larger in response to changing environments than between genomically different isolates. We demonstrate that mutations within the global regulator LasR affect more than one trait (pleiotropy) and that the interaction between mutations (epistasis) shapes the P. aeruginosa phenotypic plasticity landscape. Because of pleiotropic and epistatic effects, average genotype and phenotype measures appeared to be uncorrelated in P. aeruginosa. PMID:26126853

  3. 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

  4. 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

  5. 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.

  6. Bacteriophage-based therapy in cystic fibrosis-associated Pseudomonas aeruginosa infections: rationale and current status

    PubMed Central

    Hraiech, Sami; Brégeon, Fabienne; Rolain, Jean-Marc

    2015-01-01

    Pulmonary infections involving Pseudomonas aeruginosa are among the leading causes of the deterioration of the respiratory status of cystic fibrosis (CF) patients. The emergence of multidrug-resistant strains in such populations, favored by iterative antibiotic cures, has led to the urgent need for new therapies. Among them, bacteriophage-based therapies deserve a focus. One century of empiric use in the ex-USSR countries suggests that bacteriophages may have beneficial effects against a large range of bacterial infections. Interest in bacteriophages has recently renewed in Western countries, and the in vitro data available suggest that bacteriophage-based therapy may be of significant interest for the treatment of pulmonary infections in CF patients. Although the clinical data concerning this specific population are relatively scarce, the beginning of the first large randomized study evaluating bacteriophage-based therapy in burn infections suggests that the time has come to assess the effectiveness of this new therapy in CF P. aeruginosa pneumonia. Consequently, the aim of this review is, after a brief history, to summarize the evidence concerning bacteriophage efficacy against P. aeruginosa and, more specifically, the in vitro studies, animal models, and clinical trials targeting CF. PMID:26213462

  7. EFFECTS OF CYANOPHAGE SAM-1 UPON 'MICROCYSTIS AERUGINOSA'

    EPA Science Inventory

    Cyanophage SAM-1, which infects Synechoccus cedrorum, Anacystis nidulans and certain strains of Microcystis aeruginosa has been isolated from sewage. The host range of cyanophage SAM-1 differs from those of other reported cyanophages. Phage SAM-1 stocks are rapidly inactivated at...

  8. Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa.

    PubMed Central

    Speert, D P; Wright, S D; Silverstein, S C; Mah, B

    1988-01-01

    The phagocytic receptor for unopsonized Pseudomonas aeruginosa was characterized functionally using human monocyte-derived macrophages. Freshly isolated human peripheral blood monocytes were unable to ingest unopsonized P. aeruginosa; ingestion did not occur until the cells had been in culture for 2 d and it became maximal after 4 d. Macrophages plated on coverslips derivatized with anti-BSA IgG or with human gamma-globulin lost the capacity to phagocytose unopsonized P. aeruginosa, unopsonized zymosan, and EIgG but bound C3bi-coated erythrocytes normally. Each of the four human IgG subclasses and Fc fragments of anti-BSA IgG inhibited phagocytosis of both unopsonized P. aeruginosa and EIgG. Phagocytosis of P. aeruginosa and zymosan was markedly impaired and EIgG minimally inhibited if macrophages were plated on coverslips derivatized with mannan or when mannan was added to the phagocytosis buffer. Phagocytosis of P. aeruginosa and zymosan, and binding of EC3bi was dependent on the presence of divalent cations, but phagocytosis of EIgG was not. The macrophage phagocytic receptor for unopsonized P. aeruginosa was inactivated by proteolytic enzymes. Phagocytosis of P. aeruginosa was inhibited by D-mannose, L-fucose, and alpha methyl mannoside, but not by L-mannose, D-fucose, or D-glucose. The same sugars inhibited phagocytosis of unopsonized zymosan. We conclude that phagocytosis of unopsonized P. aeruginosa by human monocyte-derived macrophages is facilitated by mannose receptors. Images PMID:3138287

  9. Kinetics of gas-phase reactions of cyc-CF2CF2CF2CHFCH2sbnd and trans-cyc-CF2CF2CF2CHFCHFsbnd with OH radicals between 253 and 328 K

    NASA Astrophysics Data System (ADS)

    Zhang, Ni; Chen, Liang; Uchimaru, Tadafumi; Qing, Feiyao; Mizukado, Junji; Quan, Hengdao; Suda, Hiroyuki

    2015-10-01

    Rate constants for the reactions of cyc-CF2CF2CF2CHFCH2sbnd (k1) and trans-cyc-CF2CF2CF2CHFCHFsbnd (k2) with OH radicals were assessed by a relative rate method. The values were determined as (9.35 ± 5.83) × 10-13 exp[-(1197 ± 180)/T] and (8.02 ± 2.17) × 10-13 exp[-(1198 ± 80)/T] between 253 and 328 K; and (1.72 ± 0.05) × 10-14 and (1.43 ± 0.03) × 10-14 cm3 molecule-1 s-1 at 298 K, respectively. The atmospheric lifetimes were 2.8 and 3.2 years; the 100-year time horizon global warming potentials were estimated to be 211 and 241, respectively.

  10. 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

  11. The Pseudomonas aeruginosa PA01 Gene Collection

    PubMed Central

    LaBaer, Joshua; Qiu, QingQing; Anumanthan, Anukanth; Mar, Wenhong; Zuo, Dongmei; Murthy, T.V.S.; Taycher, Helen; Halleck, Allison; Hainsworth, Eugenie; Lory, Stephen; Brizuela, Leonardo

    2004-01-01

    Pseudomonas aeruginosa, a common inhabitant of soil and water, is an opportunistic pathogen of growing clinical relevance. Its genome, one of the largest among bacteria [5570 open reading frames (ORFs)] approaches that of simple eukaryotes. We have constructed a comprehensive gene collection for this organism utilizing the annotated genome of P. aeruginosa PA01 and a highly automated and laboratory information management system (LIMS)-supported production line. All the individual ORFs have been successfully PCR-amplified and cloned into a recombination-based cloning system. We have isolated and archived four independent isolates of each individual ORF. Full sequence analysis of the first isolate for one-third of the ORFs in the collection has been completed. We used two sets of genes from this repository for high-throughput expression and purification of recombinant proteins in different systems. The purified proteins have been used to set up biochemical and immunological assays directed towards characterization of histidine kinases and identification of bacterial proteins involved in the immune response of cystic fibrosis patients. This gene repository provides a powerful tool for proteome- and genome-scale research of this organism, and the strategies adopted to generate this repository serve as a model for building clone sets for other bacteria. PMID:15489342

  12. Reduced Mucosal Associated Invariant T-Cells Are Associated with Increased Disease Severity and Pseudomonas aeruginosa Infection in Cystic Fibrosis

    PubMed Central

    Smith, Daniel J.; Hill, Geoffrey R.; Bell, Scott C.; Reid, David W.

    2014-01-01

    Background Primary defects in host immune responses have been hypothesised to contribute towards an inability of subjects with cystic fibrosis (CF) to effectively clear pulmonary infections. Innate T-lymphocytes provide rapid pathogen-specific responses prior to the development of classical MHC class I and II restricted T-cell responses and are essential to the initial control of pulmonary infection. We aimed to examine the relationship between peripheral blood lymphocyte phenotype and clinical outcomes in adults with CF. Methods We studied 41 subjects with CF and 22, age matched, non-smoking healthy control subjects. Lymphocytes were extracted from peripheral blood samples and phenotyped by flow-cytometry. Lymphocyte phenotype was correlated with sputum microbiology and clinical parameters. Results In comparison to healthy control subjects, mucosal associated invariant T (MAIT)-lymphocytes were significantly reduced in the peripheral blood of subjects with CF (1.1% versus 2.0% of T-lymphocytes, P = 0.002). MAIT cell concentration was lowest in CF subjects infected with P. aeruginosa and in subjects receiving treatment for a pulmonary exacerbation. Furthermore a reduced MAIT cell concentration correlated with severity of lung disease. Conclusion Reduced numbers of MAIT cells in subjects with CF were associated with P. aeruginosa pulmonary infection, pulmonary exacerbations and more severe lung disease. These findings provide the impetus for future studies examining the utility of MAIT cells in immunotherapies and vaccine development. Longitudinal studies of MAIT cells as biomarkers of CF pulmonary infection are awaited. PMID:25296025

  13. Tobramycin Inhalation Powder (TIP): An Efficient Treatment Strategy for the Management of Chronic Pseudomonas Aeruginosa Infection in Cystic Fibrosis

    PubMed Central

    Lam, John; Vaughan, Steven; Parkins, Michael D.

    2013-01-01

    Repeated bouts of acute and chronic lung infections are responsible for progressive pulmonary function decline in individuals with cystic fibrosis (CF), ultimately leading to respiratory failure and death. Pseudomonas aeruginosa is the archetypical CF pathogen, causes chronic infection in 70% of individuals, and is associated with an accelerated clinical decline. The management of P. aeruginosa in CF has been revolutionized with the development and widespread use of inhaled antibiotics. Aerosol delivery of antimicrobial compounds in CF enables extremely high concentrations of antibiotics to be reached directly at the site of infection potentially overcoming adaptive resistance and avoiding the potential for cumulative systemic toxicities. Tobramycin inhalation powder (TIP) represents the first dry powder inhaled (DPI) antibiotic available for use in CF. DPIs are notable for a markedly reduced time for administration, ease of portability, and increased compliance. TIP has been developed as a therapeutic alternative to tobramycin inhalation solution (TIS), the standard of care for the past 20 years within CF. Relative to TIS 300 mg nebulized twice daily in on-and-off cycles of 28 days duration, TIP 112 mg twice daily via the T-326 inhaler administered on the same schedule is associated with marked time savings, increased patient satisfaction, and comparable clinical end points. TIP represents an innovative treatment strategy for those individuals with CF and holds the promise of increased patient compliance and thus the potential for improved clinical outcomes. PMID:24324354

  14. Measurement of the 250Cf component in a 252Cf neutron source at KRISS.

    PubMed

    Kim, Jungho; Park, Hyeonseo; Choi, Kil-Oung

    2014-10-01

    Neutron emission rate measurements have been carried out at the Korea Research Institute of Standards and Science using a manganese sulphate bath system for (252)Cf and (241)Am-Be sources since 2004. The relative measurement method was chosen in 2012, and the neutron emission rates agreed with those by the absolute measurement method within uncertainties. The neutron emission rate of an old (252)Cf source has been measured three times: in 2004, 2009 and 2012. The (250)Cf component was fitted to a double-exponential function of (252)Cf+(250)Cf, and the ratio of the (250)Cf component to the (252)Cf component was estimated to be 7.8 % in 2004 and 46.8 % in 2012. Underestimation of the neutron emission rates of old (252)Cf sources can be corrected if the neutron emission rate of the (250)Cf component is taken into account. PMID:24344350

  15. 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

  16. Chemotaxis by Pseudomonas aeruginosa.

    PubMed Central

    Moulton, R C; Montie, T C

    1979-01-01

    Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa. PMID:104961

  17. MexXY multidrug efflux system of Pseudomonas aeruginosa

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2012-01-01

    Anti-pseudomonas aminoglycosides, such as amikacin and tobramycin, are used in the treatment of Pseudomonas aeruginosa infections. However, their use is linked to the development of resistance. During the last decade, the MexXY multidrug efflux system has been comprehensively studied, and numerous reports of laboratory and clinical isolates have been published. This system has been increasingly recognized as one of the primary determinants of aminoglycoside resistance in P. aeruginosa. In P. aeruginosa cystic fibrosis isolates, upregulation of the pump is considered the most common mechanism of aminoglycoside resistance. Non-fermentative Gram-negative pathogens possessing very close MexXY orthologs such as Achromobacter xylosoxidans and various Burkholderia species (e.g., Burkholderia pseudomallei and B. cepacia complexes), but not B. gladioli, are intrinsically resistant to aminoglycosides. Here, we summarize the properties (e.g., discovery, mechanism, gene expression, clinical significance) of the P. aeruginosa MexXY pump and other aminoglycoside efflux pumps such as AcrD of Escherichia coli, AmrAB-OprA of B. pseudomallei, and AdeABC of Acinetobacter baumannii. MexXY inducibility of the PA5471 gene product, which is dependent on ribosome inhibition or oxidative stress, is noteworthy. Moreover, the discovery of the cognate outer membrane component (OprA) of MexXY in the multidrug-resistant clinical isolate PA7, serotype O12 deserves special attention. PMID:23233851

  18. [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

  19. LEM-CF Premixed Tool Kit

    2015-01-19

    The purpose of LEM-CF Premixed Tool Kit is to process premixed flame simulation data from the LEM-CF solver (https://fileshare.craft-tech.com/clusters/view/lem-cf) into a large-eddy simulation (LES) subgrid model database. These databases may be used with a user-defined-function (UDF) that is included in the Tool Kit. The subgrid model UDF may be used with the ANSYS FLUENT flow solver or other commercial flow solvers.

  20. Lagooning of wastewaters favors dissemination of clinically relevant Pseudomonas aeruginosa.

    PubMed

    Petit, Stéphanie M-C; Lavenir, Raphaël; Colinon-Dupuich, Céline; Boukerb, Amine M; Cholley, Pascal; Bertrand, Xavier; Freney, Jean; Doléans-Jordheim, Anne; Nazaret, Sylvie; Laurent, Frédéric; Cournoyer, Benoit

    2013-10-01

    The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds. PMID:23792168

  1. [Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

    PubMed

    Mulyukin, A L; Kozlova, A N; Sorokin, V V; Suzina, N E; Cherdyntseva, T A; Kotova, I B; Gaponov, A M; Tutel'yan, A V; El'-Registan, G I

    2015-01-01

    Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 μg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent

  2. [Susceptibility and resistence of Pseudomonas aeruginosa to antimicrobial agents].

    PubMed

    Gamero Delgado, M C; García-Mayorgas, A D; Rodríguez, F; Ibarra, A; Casal, M

    2007-06-01

    Pseudomonas aeruginosa is an opportunistic microorganism that is frequently the cause of nosocomial infections. Multiple mechanisms are involved in its natural and acquired resistance to many of the antimicrobial agents commonly used in clinical practice. The objective of this study was to assess the susceptibility and resistance patterns of P. aeruginosa strains isolated in Hospital Reina Sofia between 2000 and 2005, as well as to analyze the differences between intrahospital and extrahospital isolates in 2005 and to compare the results with those obtained in other studies. A total of 3,019 strains of P. aeruginosa from different hospitals and nonhospital settings were evaluated, taking into consideration their degree of sensitivity to different antibiotics. The MICs were determined by means of the Wider I automated system (Soria Melguizo), taking into consideration the criteria of susceptibility and resistance recommended by MENSURA. Results of the analysis showed that P. aeruginosa maintained similar levels of antimicrobial susceptibility during the period 2000-2005, with increased susceptibility to amikacin, gentamicin and tobramycin. There were also important differences in the degree of susceptibility between intrahospital and extrahospital strains, except for imipenem and fosfomycin. The intrahospital difference in susceptibility was also evaluated, emphasizing the importance of periodically studying susceptibility and resistance patterns of P. aeruginosa in each setting in order to evaluate different therapeutic guidelines, as it is not always advisable to extrapolate data from different regions. These differences can be explained by the different use of antibiotics in each center and the geographic variations of the resistance mechanisms of P. aeruginosa. PMID:17893761

  3. Adsorption of CF4 on graphite preplated with a monolayer of CF3Cl.

    PubMed

    Thomas, Petros; Velazquez, Daniel; Hess, George B

    2011-03-21

    We report a study of the adsorption of CF(4) on graphite preplated with a monolayer of CF(3)Cl, using infrared reflection absorption spectroscopy combined with ellipsometry. The saturated vapor pressure of CF(3)Cl is nearly 3 orders of magnitude smaller than that of CF(4) at the same temperature, so the main control variables are the temperature and the pressure (or chemical potential) of CF(4), together with the initial coverage of CF(3)Cl. The temperature range covered is 60-105 K. We find that, if the initial monolayer of CF(3)Cl is liquid, CF(4) continuously displaces CF(3)Cl by substitution in the monolayer. If the initial monolayer of CF(3)Cl is solid, due to either lower temperature or compression, CF(4) condenses as a second layer on the top of the CF(3)Cl layer, with only slight mixing with the original layer. This behavior persists to multiple layers of CF(4). PMID:21428651

  4. Complete genome sequence of Pseudomonas aeruginosa lytic bacteriophage PA1O which resembles temperate bacteriophage D3112.

    PubMed

    Kim, Shukho; Rahman, Marzia; Kim, Jungmin

    2012-03-01

    A novel Pseudomonas aeruginosa lytic bacteriophage (phage), PA1Ø, was isolated, and its genome was sequenced completely. This phage is able to lyse not only P. aeruginosa but also Staphylococcus aureus. Genome analysis of PA1Ø showed that it is similar to a P. aeruginosa temperate phage, D3112, with the exception of the absence of a c repressor-encoding gene, which is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa. PMID:22354942

  5. The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

    PubMed Central

    Manfredi, Pablo; Dötsch, Andreas; Blanka, Andrea; Bos, Raphael; Cornelis, Guy R.; Häussler, Susanne; Jenal, Urs

    2012-01-01

    The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed

  6. Pseudomonas aeruginosa acquisition on an intensive care unit: relationship between antibiotic selective pressure and patients' environment

    PubMed Central

    2011-01-01

    Introduction The purpose of this study was to investigate the relationship among Pseudomonas aeruginosa acquisition on the intensive care unit (ICU), environmental contamination and antibiotic selective pressure against P. aeruginosa. Methods An open, prospective cohort study was carried out in a 16-bed medical ICU where P. aeruginosa was endemic. Over a six-month period, all patients without P. aeruginosa on admission and with a length of stay >72 h were included. Throat, nasal, rectal, sputum and urine samples were taken on admission and at weekly intervals and screened for P. aeruginosa. All antibiotic treatments were recorded daily. Environmental analysis included weekly tap water specimen culture and the presence of other patients colonized with P. aeruginosa. Results A total of 126 patients were included, comprising 1,345 patient-days. Antibiotics were given to 106 patients (antibiotic selective pressure for P. aeruginosa in 39). P. aeruginosa was acquired by 20 patients (16%) and was isolated from 164/536 environmental samples (31%). Two conditions were independently associated with P. aeruginosa acquisition by multivariate analysis: (i) patients receiving ≥3 days of antibiotic selective pressure together with at least one colonized patient on the same ward on the previous day (odds ratio (OR) = 10.3 ((% confidence interval (CI): 1.8 to 57.4); P = 0.01); and (ii) presence of an invasive device (OR = 7.7 (95% CI: 2.3 to 25.7); P = 0.001). Conclusions Specific interaction between both patient colonization pressure and selective antibiotic pressure is the most relevant factor for P. aeruginosa acquisition on an ICU. This suggests that combined efforts are needed against both factors to decrease colonization with P. aeruginosa. PMID:21306623

  7. Dissemination of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania.

    PubMed

    Dortet, Laurent; Flonta, Mirela; Boudehen, Yves-Marie; Creton, Elodie; Bernabeu, Sandrine; Vogel, Anaïs; Naas, Thierry

    2015-11-01

    Fifteen carbapenemase-producing Enterobacteriaceae isolates and 12 carbapenemase-producing Pseudomonas aeruginosa isolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, Cluj-Napoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceae isolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosa isolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extended-spectrum β-lactamases and RmtC methylases. PMID:26303798

  8. Trifluoroselenoacetic acid, CF(3)C(O)SeH: preparation and properties.

    PubMed

    Gómez Castaño, Jovanny A; Romano, Rosana M; Beckers, Helmut; Willner, Helge; Della Védova, Carlos O

    2010-11-01

    The hitherto unknown trifluoroselenoacetic acid was prepared through the reaction of trifluoroacetic acid with Woollins' reagent. The compound was fully characterized by mass spectrometry, (1)H, (19)F, (77)Se, and (13)C NMR, UV-visible, IR and Raman spectroscopy, and the boiling point at 46 °C was estimated from the vapor pressure curve. An IR matrix isolation study revealed the presence of two different syn-anti and anti-syn conformers. The IR spectra of the two stereoisomers have been assigned, aided by DFT, and ab initio calculations. The UV photolysis of Ar matrix isolated CF(3)C(O)SeH yielded CO, OCSe, CF(3)SeH, and CHF(3). Apart from CF(3)SeH, these products were also obtained by vacuum flash-pyrolysis (310 °C) of gaseous CF(3)C(O)SeH. Instead of CF(3)SeH, CF(2)Se, and HF were detected among the pyrolysis products. The different decomposition pathways of CF(3)C(O)SeH are discussed. PMID:20879731

  9. Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination

    PubMed Central

    Anderson, Gregory G.; Kenney, Thomas F.; MacLeod, David L.; Henig, Noreen R.; O’Toole, George A.

    2016-01-01

    Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4 : 1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L−1, while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L−1 FTI and 16 mg L−1 tobramycin. Fosfomycin (1024 mg L−1) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L−1) and tobramycin (4 mg L−1) and high concentrations of fosfomycin (1024 mg L−1) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides. PMID:23620118

  10. Effects of Chinese medicinal herbs on a rat model of chronic Pseudomonas aeruginosa lung infection.

    PubMed

    Song, Z; Johansen, H K; Moser, C; Høiby, N

    1996-05-01

    The aim of the study was to evaluate the effects of two kinds of Chinese medicinal herbs, Isatis tinctoria L (ITL) and Daphne giraldii Nitsche (DGN), on a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF). Compared to the control group, both drugs were able to reduce the incidence of lung abscess (p < 0.05) and to decrease the severity of the macroscopic pathology in lungs (p < 0.05). In the great majority of the rats, the herbs altered the inflammatory response in the lungs from an acute type inflammation, dominated by polymorphonuclear leukocytes (PMN), to a chronic type inflammation, dominated by mononuclear leukocytes (MN). DGN also improved the clearance of P. aeruginosa from the lungs (p < 0.03) compared with the control group. There were no significant differences between the control group and the two herbal groups with regard to serum IgG and IgA anti-P. aeruginosa sonicate antibodies. However, the IgM concentration in the ITL group was significantly lower than in the control group (p < 0.03). These results suggest that the two medicinal herbs might be helpful to CF patients with chronic P. aeruginosa lung infection, DGN being the most favorable. PMID:8703440

  11. 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

  12. The Pseudomonas aeruginosa universal stress protein PA4352 is essential for surviving anaerobic energy stress.

    PubMed

    Boes, Nelli; Schreiber, Kerstin; Härtig, Elisabeth; Jaensch, Lothar; Schobert, Max

    2006-09-01

    During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection. PMID:16952944

  13. Effects of growth conditions on the production of neurotoxin 2,4-diaminobutyric acid (DAB) in Microcystis aeruginosa and its universal presence in diverse cyanobacteria isolated from freshwater in China.

    PubMed

    Fan, Hua; Qiu, Jiangbing; Fan, Lin; Li, Aifeng

    2015-04-01

    Neurotoxins β-N-methylamino-L-alanine (BMAA) and its isomer 2,4-diaminobutyric acid (DAB) have been reported previously in diverse strains of cyanobacteria. In this study, BMAA and DAB were analyzed for two strains of Microcystis aeruginosa incubated with four different levels of phosphate, nitrate, illumination, and temperature, respectively, in order to explore the effects of growth factors on toxin-producing ability of cyanobacteria. Both toxins were also screened in 17 cyanobacterial strains cultured with BG-11 medium and conventional illumination and temperature conditions, and in three field phytoplankton samples collected from different lakes in China. All samples were analyzed using a liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) system coupled with a hydrophilic interaction liquid chromatography (HILIC) column. Results showed that no BMAA was detected in any of the cyanobacterial strains grown under our laboratory culture conditions, or in any of the field samples. Production of DAB in M. aeruginosa was significantly enhanced by extreme concentrations of nutrient and physical factors. Various concentrations of DAB were also present in most cultured samples (13 of 17) of cyanobacteria and were not species specific. This is the first time to report the production of DAB in M. aeruginosa cultured under alterative conditions in laboratory. Occurrence of DAB in most of the strains examined here means that consideration should be given to the presence of this compound in freshwater environment in China. PMID:25354443

  14. CF6-6D engine performance deterioration

    NASA Technical Reports Server (NTRS)

    Wulf, R. H.; Kramer, W. H.; Pass, J. E.; Smith, J. J.

    1980-01-01

    Cruise cockpit recordings and test cell performance data in conjunction with hardware inspection data from airline overhaul shops were analyzed to define the extent and magnitude of performance deterioration of the General Electric CF6-6D model engine. These studies successfully isolated short-term deterioration from the longer term, and defined areas where a significant reduction in aircraft energy requirements for the 1980's can be realized. Unrestored losses which remain after engine refurbishment represent over 70% of the loss at engine shop visit. Sixty-three percent of the unrestored losses are cost-effective to restore which could reduce fuel consumed by CF6-6D engines in 1980 by 10.9 million gallons.

  15. Management of chronic rhinosinusitis in CF.

    PubMed

    Mainz, Jochen G; Koitschev, Assen

    2009-06-01

    Routine CF management often does not include upper airway (UAW) assessment although CFTR defects equally affect the sinonasal mucosa. Up to 50% of CF patients have chronic rhinosinusitis (CRS) and/or nasal polyps, and almost 100% reveal UAW abnormalities on CT scan. CRS impairs quality of life. UAW dysfunction in filtering, humidifying, and warming inspired air affects lower airways and the UAW is a potential site of first colonization and a reservoir for opportunistic bacteria. Therefore, UAW pathology substantially affects overall health in CF. Standard treatments are scarce and mostly lack evidence. Nasal douche can remove mucus and crusts. Recently, delivery of dornase alfa using a vibrating aerosol has shown potential as treatment for CF-related CRS. Surgery is indicated when conservative approaches fail but postoperative relapse is frequent. In summary, upper airway involvement in CF is undertreated and requires prospective investigation and an interdisciplinary consensus on diagnosis and therapy. PMID:19460681

  16. <