ENCAPSULATED PSEUDOMONAS AERUGINOSA (PSEUDOMONAS AERUGINOSA MUCOSUS) STRAINS.
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Culture Media↗
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Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida.
Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa. To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined. The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.
Bacterial Outer Membrane Proteins↗
Probing of Pseudomonas aeruginosa, Pseudomonas aureofaciens, Burkholderia (Pseudomonas) cepacia, Pseudomonas fluorescens, and Pseudomonas putida with the ferripyochelin receptor A gene and the synthesis of pyochelin in Pseudomonas aureofaciens, Pseudomonas fluorescens, and Pseudomonas putida.
The ferripyochelin receptor A (fptA) gene codes for the transport of the ferrisiderophore ferripyochelin in Pseudomonas aeruginosa. A P. aeruginosa fptA internal fragment was used to probe chromosomal DNA from P. aureofaciens, B. cepacia, P. fluorescens, P. putida, and five strains of P.aeruginosa. These bacteria all contained DNA that hybridized to the fptA fragment. Four of the five P. aeruginosa strains displayed marked and identical patterns, indicating a high degree of sequence similarities among these strains. DNA from the non-P. aeruginosa bacteria, in contrast, hybridized less to the fptA fragment. Pseudomonas aeruginosa and B. cepacia synthesize pyochelin. Experiments were performed to confirm P. fluorescens pyochelin synthesis and to determine if pyochelin, cepabactin or salicylic acid were made by P. aureofaciens, P. putida, and P. fluorescens. Only pyochelin was isolated and identified from P. fluorescens. P. aureofaciens and P. putida produced none of these compounds. While all of these bacteria contain chromosomal DNA that hybridized to the fptA fragment probe, pyochelin synthesis did not occur in all, indicating that fptA fragment hybridization cannot always be correlated with pyochelin biosynthesis.
Bacterial Outer Membrane Proteins↗
Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli.
R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.
Arginine↗
[Evaluation of culture media used in the differentiation of Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, isolated from clinical human material].
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Culture Media↗
[Clinical study on nosocomial infection of Pseudomonas aeruginosa. II. Pseudomonas aeruginosa in the feces and urinary Pseudomonas aeruginosa infection].
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Cross Infection↗
Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa.
Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.
Antibodies, Bacterial↗
[Method and result of oxygen addition in the culture process of Pseudomonas aeruginosa].
Pseudomonas aeruginosa septicemia are frequent complications of patients with major burns. To evaluate more rapid culture methods for diagnosis, we have established a new culture method of pseudomonas aeruginosa by oxygen addition. The results of vitro study showed that the growth time of pseudomonas aeruginosa was 8 to 12 hours earlier by the new culture method than by the ordinary method, when pseudomonas aeruginosa were incubated in broth at 37 degrees C to achieve approximately 50 to 65 colony-forming units/bottle. A total of 60 samples was tested when each culture bottle was added approximately 2 to 10 colony-forming units. The percentage of positive culture was 75% by venting oxygen addition method and 33.3% by ordinary method. The result of assay of 26 blood samples from patients with major burns showed that the percentage of positive blood cultures was 69.2% by the new method and 42.3% by the ordinary method. The culture time of the new method was 4 to 12 hours earlier than the ordinary method. This technique may lead to a greater chance of recovery and rapid detection of pseudomonas aeruginosa from blood samples.
Bacteriological Techniques↗
[Severe lesions of the petrous bone caused by pseudomonas aeruginosa].
Pseudomonas aeruginosa is often isolated in infections of the ear cleft. In some circumstances, this organism can cause serious petrous or peri-petrous lesions. Two pictures are seen: Malignant external otitis with severe headaches, signs of external otitis, and usually pseudomonas aeruginosa is isolated. This is usually seen in an elderly diabetic patient. Nerve paralysis is the main risk. The other complications, very grave in the past, are rare nowadays with the use of selective antibiotic treatment. Pseudomonas aeruginosa is also the causative organism in extensive osteitis of the skull base. Diagnostic problems are seen in case of specific infections or tumoral lesions. The treatment includes the same medications as for the malignant external otitis, as well as complete surgical excision.
Aged↗
Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa.
Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.
Animals↗
Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa.
Pseudomonas aeruginosa cytotoxin is a protein toxin exhibiting cytotoxic effects on various eukaryotic cells. The toxin was purified from a crude extract of Pseudomonas aeruginosa 158 by trypsin treatment and serial chromatography. The purified cytotoxin was electrophoresed as a single protein band on polyacrylamide gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS). The molecular weight of the purified toxin was determined to be 29,000 by SDS-PAGE and gel filtration chromatography in the presence of 6 M guanidine hydrochloride; the isoelectoric point was pH 6.0. The N-terminal amino acid sequence of the purified toxin was determined. The purified toxin showed the strongest cytotoxic effect on rabbit polymorphonuclear leukocytes and diverse degrees of cytotoxic effects on various eukaryotic cells including red blood cells and cultured cells.
Amino Acid Sequence↗
Ultraviolet-B lethal damage on Pseudomonas aeruginosa.
Pseudomonas aeruginosa has shown an increased sensitivity compared with that of Escherichia coli and Enterobacter cloacae, when they were exposed to 0.4 kJ/m2 of ultraviolet-B radiation. The rapid decay in cell viability observed in Pseudomonas aeruginosa after the irradiation was influenced by factors such as culture media and the presence of pyocyanine during the irradiation. The radioinduced lethal damage could be prevented by photoreactivating treatment, indicating that pyrimidine dimer formation was the mechanism causing bacterial death. The results indicate that several environmental conditions may act as protective agents against ultraviolet-B-induced damage.
Cell Death↗
Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa.
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.
California↗
The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa.
Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. Research has shown that the C-terminal region of the pilin monomer contains the epithelial cell binding domain, which is semiconserved in seven different strains of this bacterium. Antibodies to this region of the pilin molecule are also able to block and prevent the infection process. As there is a degree of sequence and structural homology in the C-terminal region and all strains examined have been shown to bind to the same cell surface receptor, we reasoned that it should be possible to produce a synthetic peptide consensus sequence which would provide cross-reactive antiserum from a single peptide immunogen inhibiting the adherence of the known strains of P. aeruginosa. In this article we examine the cross-reactivity of five rabbit polyclonal antisera. One has been raised against the cell-surface receptor binding domain of native PAK strain pilin (residues 128-144) while the others have been raised to analogues of this region. Analysis of the cross-reactivity of these antisera, using competitive ELISA assay, has shown that it is possible to manipulate the amino acid sequence of a peptide immunogen to generate antiserum, which exhibits enhanced cross-reactivity to various strains of P. aeruginosa. Furthermore, when this peptide is conjugated to tetanus toxoid and used to vaccinate mice it provided cross-reactive protection against heterologous challenge with PAO strain bacteria. The results of these experiments are analyzed, and the applicability of our hypothesis and the implications of this approach to the design of a strain-independent consensus vaccine for immunization against Pseudomonas aeruginosa are discussed.
Amino Acid Sequence↗
Effects of sublethal concentrations of benzylpenicillin on Pseudomonas aeruginosa.
Pseudomonas aeruginosa produces a low basal level of beta-lactamase (0.002 to 0.004 IU/mg of protein when benzylpenicillin is used as substrate). The beta-lactamase specific activity can be increased several hundredfold by growing the bacteria in the presence of beta-lactam antibiotics. This induction was studied in Pseudomonas aeruginosa 1822s. The single-cell resistance to benzylpenicillin was 750 mug/ml. In liquid culture all concentrations of benzylpenicillin tested (25 to 2,000 mug/ml) affected the bacteria similarly: beta-lactamase formation was induced, the cells became cholate sensitive, growth rate decreased, filaments were formed, and beta-lactamase was excreted. The effect appeared earlier the higher the concentration of the antibiotic. Most of the effects obtained are concerned with the functioning of the outer membrane. The excretion of beta-lactamase seems to be due to an opening of the periplasmic volume rather than to lysis of the cells. Carbenicillin gave the same effects as benzylpenicillin at the same concentrations; the 10-fold lower resistance to carbenicillin than to benzylpenicillin can be explained by the inability of the inducible beta-lactamase to hydrolyze carbenicillin. The induced beta-lactamase was first cell bound and to a great extent located in the periplasmic volume, but later it was excreted into the medium. This extracellular activity was responsible for the detoxification of the medium. This is analogous to the behavior of gram-positive bacteria rather than to that of Enterobacteriaceae.
Carbenicillin↗
[Heat-stable hemolysin of Pseudomonas aeruginosa].
Pseudomonas aeruginosa produces at least two distinct hemolysins: heat-labile hemolysin, which is known as phospholipase C, and heat-stable hemolysin, which is considered to be a glycolipid but its true nature is not found yet. Cellophane agar plate method was applied to hasten and enhance the hemolysin production by Pseudomonas aeruginosa, and the hemolysin obtained by this method was examined biochemically. This heat-stable hemolysin consisted of two major acidic glycolipids. One was considered to be the same glycolipid as that obtained by the same method of Jarvis and Johnson's from the peptone-glycerol culture filtrate of the same organism, which is composed of two moles of rhamnose and two moles of beta-hydroxy decanoic acid, and the other one being a glycolipid composed of one mole of rhamnose and two moles of beta-hydroxy decanoic acid. By this method, the rhamnolipid with one mole of rhamnose was produced more than the rhamnolipid with two moles of rhamnose, and they were produced in the same ratio from the first to fifth culture day. From the examinations of hemolytic activities of the hydrolyzed products of these glycolipids, the hemolysis producing moiety of the hemolysin was considered to be the dimer of beta-hydroxy decanoic acid contained in the glycolipid molecule. Hemolytic activities of these glycolipids are regarded as their detergent like effects. Since these activities are interfered by small amount of serum protein, their pathogenic role would not be important in vitro. However, their interaction with other toxic substances produced by this organism would be subject to be solved.
Bacteriological Techniques↗
[Effects of azithromycin on the virulence of Pseudomonas aeruginosa].
Pseudomonas aeruginosa is a saprophyte opportunistic bacteria which frequently colonizes the respiratory tract of patients presenting a severe chronic bronchitis pathology. Secreting a number of exotoxins and enzymes inducing an inflammation and necrosing of the surrounding tissues, it provokes irreversible pulmonary lesions. Different experimental in vitro works evidenced macrolides activity on the production and/or secretion of these factors, with a diminution of elastase, protease, lecithinase and D-nase synthesis. Among the macrolides, azithromycin seems to have the most pronounced activity. In vivo, some patients suffering from bronchiolitis or cystic fibrosis have been clinically improved with a treatment using erythromycin, or clarithromycin or azithromycin. These very preliminary results demand to be confirmed but the macrolides could allow a decrease of Pseudomonas aeruginosa pathogenicity and thus stop the deterioration of pulmonary functions.
Anti-Bacterial Agents↗
Amino acid-mediated induction of the basic amino acid-specific outer membrane porin OprD from Pseudomonas aeruginosa.
Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.
Amino Acids↗