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PubMed1994

A multicenter comparison of methods for typing strains of Pseudomonas aeruginosa predominantly from patients with cystic fibrosis. The International Pseudomonas aeruginosa Typing Study Group.

Typing methods in seven laboratories were compared for their ability to type reproducibly 200 strains of Pseudomonas aeruginosa, predominantly from patients with cystic fibrosis (CF). Methods included lipopolysaccharide (LPS) serotyping, phage susceptibility typing, bacteriocin production, pilin gene typing, and analysis by restriction fragment length polymorphism (RFLP) with a probe upstream from the exotoxin A gene. The methods differed substantially in their capacity to identify unique typing patterns and to type each strain reproducibly on three occasions. For strains from patients with CF, the RFLP typing method had the greatest discriminatory power (30 unique patterns, 105 [70%] of 150 typed reproducibly). LPS serotyping appeared to be equivalent to RFLP typing for discriminating among P. aeruginosa strains from the environment and from clinical sources other than CF patients. RFLP analysis appears to be the best method for typing P. aeruginosa strains with rough LPS (such as from patients with CF). LPS serotyping appears preferable for other indications as it is simpler to perform.

ADP Ribose Transferases↗
PubMedDate not supplied

Combined host and specific anti-Pseudomonas-directed therapy for Pseudomonas aeruginosa infections in burned mice: experimental results and theoretic considerations.

Four strains of Pseudomonas aeruginosa showed significant variation in the elaboration of the virulence-associated exoproducts, exotoxin A and proteases. These variations appeared to affect the mortality observed when these strains were used to infect burned mice. Further, these variations seemed to influence the efficacy of individual after sepsis treatments designed to (1) reduce total microbial load in the host (HIg treatment), (2) reduce total circulating protease load in the host (alpha 1-PI treatment), or (3) neutralize the specific effects of exotoxin A (antitoxin treatment). Combined treatment, using all of these approaches, was significantly better than individual treatment.

ADP Ribose Transferases↗
PubMed2000

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

ADP Ribose Transferases↗
PubMed1981

Catabolism of Naphthalenesulfonic Acids by Pseudomonas sp. A3 and Pseudomonas sp. C22.

Naphthalene and two naphthalenesulfonic acids were degraded by Pseudomonas sp. A3 and Pseudomonas sp. C22 by the same enzymes. Gentisate is a major metabolite. Catabolic activities for naphthalene, 1-naphthalenesulfonic acid, and 2-naphthalenesulfonic acid are induced by growth with naphthalene, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, methylnaphthalene, or salicylate. Gentisate is also an inducer in strain A3. Inhibition kinetics show that naphthalene and substituted naphthalenes are hydroxylated by the same naphthalene dioxygenase. Substrates with nondissociable substituents such as CH(3), OCH(3), Cl, or NO(2) are hydroxylated in the 7,8-position, and 4-substituted salicylates are accumulated. If CO(2)H, CH(2)CO(2)H, or SO(3)H are substituents, hydroxylation occurs with high regioselectivity in the 1,2-position. Thus, 1,2-dihydroxy-1,2-dihydronaphthalene-2-carboxylic acids are formed quantitatively from the corresponding naphthalenecarboxylic acids. Utilization of naphthalenesulfonic acids proceeds by the same regioselective 1,2-dioxygenation which labilizes the C-SO(3) bond and eliminates sulfite.

Journal Article↗
PubMed1984

Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C.

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.

Journal Article↗
PubMed1985

Enhancement of Pyrroloquinoline Quinone Production and Polyvinyl Alcohol Degradation in Mixed Continuous Cultures of Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C with Mixed Carbon Sources.

In a mixed continuous culture of Pseudomonas putida VM15A and Pseudomonas sp. strain VM15C with polyvinyl alcohol (PVA) as the sole source of carbon, growth of the PVA-degrading bacterium VM15C and, hence, PVA degradation were limited by the growth factor, pyrroloquinoline quinone, produced by VM15A. Feeding of a carbon source for VM15A, ethanol, with PVA enhanced pyrroloquinoline quinone production and caused increases in the VM15C population and PVA degradation in a mixed continuous culture. There was an optimum range for PVA degradation of the ethanol concentration, although pyrroloquinoline quinone concentrations in continuous mixed cultures increased with increasing ethanol concentration.

Journal Article↗
PubMed1988

Heat Production by the Denitrifying Bacterium Pseudomonas fluorescens and the Dissimilatory Ammonium-Producing Bacterium Pseudomonas putrefaciens during Anaerobic Growth with Nitrate as the Electron Acceptor.

The heat production rate and the simultaneous nitrate consumption and production and consumption of nitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitrate reducers. Pseudomonas fluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitrite or nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption and decreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpy change value of -800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrate consumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatory ammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitrite consumption commenced after depletion of the nitrate; consequently, two phases were noted in the heat production rate curve during growth. A mean experimental enthalpy change value of -810 kJ/mol of nitrate was obtained for different concentrations of nitrate.

Journal Article↗
PubMed1994

Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14.

Changes in culturability and outer membrane protein profiles were investigated in Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains.

Journal Article↗
PubMed1995

Survival and Plant Growth Promotion of Detergent-Adapted Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7NSK2.

Four detergents were tested as selective C sources for the plant growth-promoting rhizobacteria Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15. CO-720 (Igepal CO-720) or DOS (dioctyl sulfosuccinate), applied at 0.2% to the soil, increased the number of detergent-adapted, inoculated strains by almost 1.5 log units after 25 days, accounting for virtually the entire increase in total bacteria. The same dose of Tween 80 or N-laurylsarcosine, on the other hand, increased the indigenous populations by almost 2.5 log units, with only minor increases in the number of detergent-adapted inoculated strains. When CO-720 or DOS was initially supplied, the number of detergent-adapted 7NSK2 organisms was about 2 log units higher after 3 months of incubation than for the detergent-unadapted strain. This better survival resulted in a significantly higher root colonization of maize in a pot experiment with soil inoculation, with a significantly (P <= 0.05) higher shoot dry weight (18 to 33%). In a first field experiment with rhizosphere inoculation of 1-month-old maize plants, no effects on the height of two maize cultivars could be observed 1 month after inoculation. In a second field experiment, leaf and stem dry weights of yellow mustard and grass dry weight were increased in the treatments with seed and soil inoculation of the detergent-adapted 7NSK2 in combination with CO-720 application by, respectively, 7 to 8%, 19 to 23%, and 20 to 31%, although only the increases in grass dry weight were statistically significant at P <= 0.1. To some extent, 7NSK2 and DOS application also positively affected the mineral content of yellow mustard.

Journal Article↗
PubMed1995

Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy.

In Pseudomonas aeruginosa, the transcriptional activator LasR and the Pseudomonas autoinducer PAI, are necessary for efficient transcriptional activation of the lasB gene, encoding elastase (L. Passador, J. M. Cook, M.J. Gambello, L. Rust, and B. H. Iglewski, Science 260:1127-1130, 1993). The transcriptional start points of lasI in Escherichia coli and P. aeruginosa were determined by S1 nuclease mapping. In the presence of both LasR and PAI, the start site, T1, is located at position -25 relative to the ATG translational start codon. A minor transcriptional start, T2, is found at position -13 when lasI is transcribed in the absence of either LasR or PAI in P. aeruginosa and E. coli, respectively. To begin to closely examine the regulation of lasI, whose product is involved in the synthesis of PAI, a lasI-lacZ fusion on a lambda phage was constructed to form monolysogens of E. coli MG4. Lysogens supplied only with either lasI or lasR via multicopy plasmids demonstrated no significant increase in beta-galactosidase expression compared with control levels. Lysogens in which both lasR and lasI were supplied in multicopy exhibited a 62-fold increase in expression, and a lysogen in which lasR was supplied in trans and which was grown in the presence of exogenous PAI exhibited a 60-fold increase. Thus, LasR and PAI are necessary for the full expression of lasI in E. coli. The interchangeability of the P. aeruginosa and Vibrio fischeri homologs LasR and LuxR and their respective autoinducers, PAI and VAI, as activators of lasI-lacZ was examined. Only the combination of LasR and PAI significantly increased the expression of lasI. The comparison of lasI-lacZ and lasB-lacZ expression lysogens grown in the presence of lasR and PAI revealed that half-maximal expression of lasI required 0.1 nM PAI, in contrast to the 1.0 nM PAI necessary for lasB half-maximal expression. These results suggest an autoinduction regulatory hierarchy in which LasR and low PAI concentrations primarily activate lasI expression in a regulatory loop. With the accumulation of PAI, secondary activation of virulence product genes such as lasB occurs.

Bacterial Proteins↗
PubMed1982

Evidence for Pseudomonas antigen in immune complexes in Pseudomonas osteomyelitis.

In infections associated with immune complex disease, microbial antigens have rarely been found in the complexes. Using an enzyme-linked immunoassay, we studied the immune complexes of a patient who had hematogenous Pseudomonas aeruginosa osteomyelitis associated with palpable purpura, arthritis, and microscopic hematuria. After separation of the complexes into high and low molecular weight fractions, a 6-fold selective concentration of P aeruginosa antibody was found in the low molecular weight fraction compared with the concentration of the serum. Following disruption of immunoglobulin, the high molecular weight fraction competed with solid-phase P aeruginosa antigen for P aeruginosa antibody from another source. After successful treatment of the infection, the patient's symptoms resolved, and the complexes disappeared. These findings strongly suggest that immune complexes in this patient contained P aeruginosa antigen and antibody that may have been pathogenetic in his disease.

Antibodies↗
PubMed1997

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

ADP Ribose Transferases↗
PubMed1985

Shuttle vector for Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa.

A hybrid plasmid capable of replication in 2 different genera, Escherichia and Pseudomonas, was constructed. This plasmid DNA can be used as a cloning vector in E. coli and pseudomonades cells. The described hybrid plasmid pLD411 has been constructed on the basis of 2 small E. coli vector R-plasmids used in our laboratory and cryptic plasmid pWW2 or P. putida MT1. Plasmid pLD411 DNA was mapped with restrictases; its biological activity in transformations of different bacterial strains was studied, and the characteristics of transformed cells were also described.

Cloning, Molecular↗
PubMed1999

Pseudomonas aeruginosa phage lysate as an immunobiological agent. 1. Selection of Pseudomonas aeruginosa clinical strains for phage lysate preparation.

A total of 2087 Pseudomonas aeruginosa isolates collected during the period 1994-1997 were used as starting material. Out of 1704 in-patient isolates, 299 strains were selected for the preparation of phage lysates but only five strains provided stable lysates, i.e., maintained the ability to be repeatedly and completely lysed by the appropriate phage in the course of several years. A set of 193 out-patients (189) and water sources (4) isolates failed to yield strains suitable for phage lysate preparation; 190 strains isolated abroad from patients with cystic fibrosis or respiratory infections included three isolates which, despite having a high degree of mucus production, were suitable for lysate preparation. The antigenic pattern of the phage lysates was ascertained by SDS-polyacrylamide gel electrophoresis.

Antigens, Bacterial↗
PubMed1994

Protection of immunosuppressed mice against translocation of Pseudomonas aeruginosa from the gut by oral immunization with recombinant Pseudomonas aeruginosa outer membrane protein I expressing Salmonella dublin.

AroA Salmonella dublin was used as recipient for a plasmid coding for the outer membrane protein I (OprI) of Pseudomonas aeruginosa. Oral immunization of Balb/c mice with recombinant S. dublin induced serum IgG and IgA antibodies against P. aeruginosa. In spleen and Peyer's patches anti-P. aeruginosa IgG- and IgA-secreting cells could be measured by the ELISPOT technique. In an oral challenge of immunosuppressed mice with P. aeruginosa the orally immunized animals had a 58-fold higher LD50 than control animals.

Administration, Oral↗
PubMed2004

Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains.

The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P. fluorescens were sequenced. ITS1 exhibited significant sequence variability among different operons within a single genome. From 1 to 4 types of ITS1 were found in individual genomes of the P. syringae and P. fluorescens strains. A total of eight ITS1 types were identified among strains studied. The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks. The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks. The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

Base Sequence↗
PubMed2006

Early rise of anti-pseudomonas antibodies and a mucoid phenotype of pseudomonas aeruginosa are risk factors for development of chronic lung infection--a case control study.

P. aeruginosa is the most significant pathogen in CF lung disease. Chronic infection is preceded by a period of intermittent colonization. Early aggressive antimicrobial treatment at initial detection of P. aeruginosa in lower respiratory tract (LRT) secretions can prevent transition to chronic infection in approximately 80% of the patients, while the rest progress to chronic infection in spite of treatment. To analyze risk factors for development of chronic infection, a cohort of 89 CF patients free of chronic infection at the study period start was followed for 10 years. 28 of the patients (study group) developed chronic infection in spite of early treatment and 28 age-matched patients who did not (controls) were included in the analysis. During the 3 years period prior to onset of chronic infection, P. aeruginosa-positive cultures were more frequent in the study group than in the controls (2.2 vs. 0.5 per year, p<0.0001). Growth of mucoid strains of P. aeruginosa was more frequent in study group than in controls (11.5% vs. 0%, p<0.0001). Most important, specific anti-pseudomonal IgG serum antibodies were significantly higher in the study group than in controls (0.98 Elisa Units vs. 0.53, p=0.04) already 3 years prior to onset of chronic infection and increased 0.44 EU pr year in the study group but remained at the initial level in the control group (p<0.005). Occurrence of Aspergillus-positive cultures were significantly more frequent in the study group than in controls (p=0.01). The strongest risk factor for development of chronic P. aeruginosa infection was increasing levels of specific anti-pseudomonal antibodies, specifically of IgG1 and IgG4 subclass and total anti-Pseudomonas IgG, 3 years prior to onset of chronic infection, with odds ratio (OR) 8.9, 7.7 and 7.4, respectively (p<0.005), and growth of mucoid P. aeruginosa strains with OR of 7.4, p=0.006). Occurrence of Aspergillus was also a risk factor for developing chronic P. aeruginosa infection with the OR of 4.7 (p=0.008).

Adolescent↗
PubMed1995

Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis.

Patients with cystic fibrosis (CF; N = 26) and with no prior history of infection with Pseudomonas aeruginosa were immunized with an octavalent O-polysaccharide-toxin A conjugate vaccine. During the next 4 years, 16 patients (61.5%) remained free of infection and 10 (38.5%) became infected. Total serum antilipopolysaccharide (LPS) antibody levels induced by immunization were comparable in infected and noninfected patients. In contrast, 12 of 16 noninfected versus 3 of 10 infected patients (p = 0.024) mounted and maintained a high-affinity anti-LPS antibody response. When compared retrospectively with the rate in a group of age- and gender-matched, nonimmunized, noncolonized patients with CF, the rate at which P. aeruginosa infections were acquired was significantly lower (p < or = 0.02) among all immunized versus nonimmunized patients during the first 2 years of observation. Subsequently, only those immunized patients who maintained a high-affinity anti-LPS antibody response had a significant reduction (p < or = 0.014) in the rate of infection during years 3 and 4. Smooth, typeable strains of P. aeruginosa predominated among immunized patients; rough, nontypeable strains were most frequently isolated from nonimmunized patients. Mucoid variants were isolated from one immunized patient versus six nonimmunized patients. These results indicate that the induction of a high-affinity P. aeruginosa anti-LPS antibody response can influence the rate of infection in patients with CF.

Adolescent↗