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Experimental Pseudomonas infection in mice: acquired resistance against Pseudomonas septicemia and altered susceptibility in BCG infected mice.

Pseudomonas septicemia in mice caused the early death in the first three days of infection without any localized lesions. Localized lesions such as abscess are observed in the kidney and liver after the third day of infection. The early death increased in the BCG infected mice showing an increased level of macrophage activation. It seemed that such early death is due to endotoxin produced by Pseudomonas aeruginosa. However, the BCG infected mice showing an enhanced antibody formation were more resistant to pseudomonas septicemia. Immune serum protected such death, but immune spleen cells did not. Furthermore immune serum also protected the increased death in BCG infected mice.

Animals↗

Pseudomonas sepsis does not cause more severe cardiovascular dysfunction in patients than non-Pseudomonas sepsis.

To evaluate the clinical relevance of the experimental findings of a more severe cardiac depression in Pseudomonas (P.) than in non-P. sepsis, we retrospectively compared the hemodynamic data in 26 patients with P. sepsis (20 cases, single pathogen; six cases, more positive cultures with P. than with non-P. species), and 102 with non-P. sepsis. As in other studies, the left ventricular stroke work index (LVSWI) was used to assess cardiac performance. The two groups (all numbers are means) had a similar disease and sepsis severity profile (P. vs. non-P: septic shock, 81% vs. 87%; APACHE II scores, 29.1 vs. 29.2; Elebute sepsis scores, 18.1 vs. 18.1; mortality, 58% vs. 62%). Preload (pulmonary capillary wedge pressure 15.0 vs. 16.3 mm Hg) and systemic vascular resistance (588 vs 572 dyn.cm-5.sec) were comparable. Cardiac performance displayed no significant difference (LVSWI, 42.8 vs. 38.3 g.m/m2), a result reproduced in the subgroups with culture-proven bacteremia, with or without preexisting cardiovascular disease or septic shock. Thus, our data suggest that there is no difference in the degree of cardiovascular dysfunction in patients with Pseudomonas compared to non-Pseudomonas sepsis of otherwise equivalent disease severity.

Cardiovascular System↗

Serum IgG antibody to outer membrane antigens of Pseudomonas cepacia and Pseudomonas aeruginosa in cystic fibrosis.

IgG antibody to the outer membrane of Pseudomonas cepacia was quantified in sera from controls without cystic fibrosis (CF) and from Pseudomonas aeruginosa- and P. cepacia-infected and noninfected patients with CF. The mean antibody titer in the P. cepacia-infected group was significantly higher than that in the other three groups; the mean titer in the P. aeruginosa-infected group was significantly higher than that in the noninfected and control groups. Preabsorption of CF sera with P. cepacia outer membrane produced a significantly lower mean antibody concentration than did matched samples preabsorbed with an equal amount of lipopolysaccharide. By western blot, significantly more P. cepacia-infected patients produced IgG to the 27- than the 36-kilodalton (kDa) porin protein of P. cepacia; 12 of 16 P. aeruginosa-infected patients produced IgG to the 27-kDa porin. Sera from all patients in both groups contained IgG to the porin protein of P. aeruginosa by western blot. We conclude that the 27-kDa porin of P. cepacia is antigenic in most P. cepacia-infected patients with CF and that some P. cepacia outer membrane components may be antigenically related to those of P. aeruginosa.

Adolescent↗

Daytime orthokeratology associated with infectious keratitis by multiple gram-negative bacilli: Burkholderia cepacia, Pseudomonas putida, and Pseudomonas aeruginosa.

PURPOSE: To report a case of a corneal ulcer in a patient who wore orthokeratology contact lenses during the day. METHODS: Case report. RESULTS: A 16-year-old girl who underwent orthokeratology treatment developed a corneal ulcer in her right eye. The refractive status of the affected eye was -5.75 -1.75 x 180, and the best-corrected visual acuity was 20/40. Corneal topography showed the temporal upper dislocation of a central flattened zone in the right eye. The patient had worn orthokeratology contact lenses during the day for more than 4 years. Her ulcer became worse after treatment with tobramycin and gentamicin for 1 day. After treatment with ciprofloxacin, the ulcer healed, and visual acuity returned to 20/20 with spectacle correction. Cultures of the cornea, contact lens, and lens solution all grew Burkholderia cepacia, Pseudomonas putida, and Pseudomonas aeruginosa. CONCLUSIONS: Improper fitting of lenses and contamination of lenses or solutions in orthokeratology therapy are risk factors for a corneal ulcer, even when patients wear orthokeratology lenses only during the day.

Adolescent↗

In vitro activity of aztreonam combined with tobramycin and gentamicin against clinical isolates of Pseudomonas aeruginosa and Pseudomonas cepacia from patients with cystic fibrosis.

The in vitro activity of aztreonam combined with tobramycin and with gentamicin was assessed in 78 clinical isolates of Pseudomonas aeruginosa and 11 clinical isolates of Pseudomonas cepacia from patients with cystic fibrosis. Synergy was detected in 56.4% of P. aeruginosa isolates and 60% of P. cepacia isolates with the aztreonam-tobramycin combination and in 49.3% of P. aeruginosa isolates and 81.8% of P. cepacia isolates with the aztreonam-gentamicin combination. No antagonism was observed. These combinations merit clinical evaluation in the treatment of patients with cystic fibrosis.

Aztreonam↗

Crc is involved in catabolite repression control of the bkd operons of Pseudomonas putida and Pseudomonas aeruginosa.

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

[Spontaneous phage production in Pseudomonas pseudomallei and in a range of hosts of melioidosis phages among representatives in the genus Pseudomonas].

A capacity to spontaneous phage production has been studied in 57 melioidosis museum strains. The strains were comparatively analyzed by the levels of frequencies of spontaneous phage production. It is established that 40 of 57 studied strains of Pseudomonas pseudomallei are capable to spontaneously produce phages with frequency from 10(7) to 1. On the indicator lawn of P. mallei phages formed negative colonies of different morphology transparent, turbid and turbid with secondary growth in the centre. No distinct correlation between the frequency of spontaneous phage production and morphology of negative colonies was revealed. The range of hosts of melioidosis phages was determined among 88 representatives of Pseudomonas genus. All melioidosis strains were resistant to their own phages. Phages, identical by the spectrum of lithic effect, were not found even in the case of quantitative coincidence of sensitive strains. Maximum activity was observed in 8 lysates which were lysed 42 to 53 strains of 88 under study.

Burkholderia cepacia↗

Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82.

A 12,808-nucleotide containing DNA fragment cloned from naphthalene-utilizing (Nah+) Pseudomonas aeruginosa PaK1 was analyzed and compared with the genes (pah(OUS)) of a 14,462-nucleotide DNA fragment from Pseudomonas putida OUS82. The DNA sequence analyses demonstrated that the naphthalene upper-pathway genes and their deduced enzymes were very similar between the two bacteria: nucleotide similarities, 83-93%; amino acid similarities, 79-95%. These genes were also similar to those of the nah operon of plasmid NAH7; in particular, the OUS82 genes were similar to the nah genes, whereas the PaK1 genes were almost identical to the dox genes of Pseudomonas sp. C18. A region homologous with the 84-bp repeated sequence that Eaton (J. Bacteriol., 176, 7757-7762, 1994) has found at a site upstream of he nah operon was found only in a region downstream of the pah(PaK) gene cluster in PaK1 and on both sides of the pah(OUS) gene cluster in OUS82. A PaK1 gene, corresponding to an unknown gene (nahQ) in the nah operon, is located between the 1,2-dihydroxynaphthalene dioxygenase gene and the trans-o-hydroxybenzylindenepyruvate (tHBP A) hydratase-aldolase gene (nahE), and was suggested to be involved in the conversion of naphthalene to salicylate. Just downstream of the pah(PaK) gene cluster, a portion of a region was identical to one-third of the transposase gene (tnpA) in a phenol-catabolic plasmid pEST1226.

Journal Article↗

Substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia, with special reference to tyrosine phosphatase.

The substrate response in acid phosphatase activity of Pseudomonas pseudomallei and Pseudomonas cepacia was examined with different phosphate esters including hexose phosphates and phosphoaminoacids in a whole cell assay system. The enzymatic activity against each substrate was evaluated in terms of percent activity to that against para-nitrophenyl phosphate set as 100. A remarkable finding was that the phosphatase reaction was the highest with phosphotyrosine or phosphoserine as substrate showing 180% activity. This tyrosine phosphatase activity was resistant to heating at 60 C for 20 min and inhibited greatly by 0.1% ZnCl2. Pseudomonas cepacia showed the same pattern of substrate response and the same characteristics of tyrosine phosphatase activity.

Acid Phosphatase↗

Cloning, DNA sequencing, and amino acid sequencing of catechol 1,2-dioxygenases (pyrocatechase) from Pseudomonas putida mt-2 and Pseudomonas arvilla C-1.

Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid and is encoded by a catA gene. We have cloned a catA gene from Pseudomonas putida mt-2 using a PCR product of amino acid sequence-based primers as a probe. The amino acid sequence deduced from the 930 nucleotides was in complete agreement with the chemically determined sequence of the protein. Crude extracts of Escherichia coli cells carrying the catA gene downstream from the lac promoter showed the enzyme activity. By using the same probe, we also cloned and sequenced the catA beta gene for catechol 1,2-dioxygenase isozyme beta beta from Pseudomonas arvilla C-1, which has three isozymes, alpha alpha, alpha beta, and beta beta (C. Nakai, H. Horiike, S. Kuramitsu, H. Kagamiyama, and M. Nozaki, 1990, J. Biol. Chem. 265, 660-665). There was very high homology between isozyme beta beta of the C-1 strain and the enzyme of the mt-2 strain in both the amino acid (98%) and the DNA sequences (97%). A preference for the use of codons terminating in C and G was found in the coding region of both the enzymes, which contributed to the high G + C content (65-66%) of the genes. A comparison of the DNA sequences of various catA genes from other sources revealed their common ancestry, whereas a comparison of the amino acid sequences of the enzymes revealed clear reflection of substrate specificity. Tyrosyl and histidyl residues for proposed ligands of ferric ion are conserved in all catechol 1,2-dioxygenases.

Amino Acid Sequence↗

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 bp in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84--Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.

Amino Acid Sequence↗

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa.

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 microg/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20-40 microg/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20-100 microg/ml. Similarly, 100 microg/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).

Bacterial Proteins↗

Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains.

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.

Bacterial Proteins↗

Kinetics of BTEX biodegradation by a coculture of Pseudomonas putida and Pseudomonas fluorescens under hypoxic conditions.

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500 mg l(-1) of benzene, 600 mg l(-1) of o-xylene, and 1000 mg l(-1) of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO2 and H2O without producing any identifiable intermediate metabolites.

Aerobiosis↗

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.

Adenine↗

Electrical wiring of Pseudomonas putida and Pseudomonas fluorescens with osmium redox polymers.

Two different flexible osmium redox polymers; poly(1-vinylimidazole)12-[Os-(4,4'-dimethyl-2,2'-di'pyridyl)2Cl2](2+/+) (osmium redox polymer I) and poly(vinylpyridine)-[Os-(N,N'-methylated-2,2'-biimidazole)3](2+/3+) (osmium redox polymer II) were investigated for their ability to efficiently "wire" Pseudomonas putida ATCC 126633 and Pseudomonas fluorescens (P. putida DSM 6521), which are well-known phenol degrading organisms, when entrapped onto cysteamine modified gold electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os(2+/3+)-functionalities are located. The bacterial cells were adapted to grow in the presence of phenol as the sole source of organic carbon. The performance of the redox polymers as mediators was investigated for making microbial sensors. The analytical characteristics of the microbial sensors were evaluated for determination of catechol, phenol and glucose as substrates in both batch analysis and flow analysis mode.

2,2'-Dipyridyl↗

Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa.

Regulation of medium-chain-length polyhydroxyalkanoate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida was studied conducting PHA accumulation experiments and transcriptional analysis of PHA biosynthesis genes with wild type strains and rpoN-negative mutants. In P. putida PHA accumulation was RpoN-independent, whereas in P. aeruginosa PHA accumulation was RpoN-dependent. Transcriptional analysis applying reverse transcriptase-polymerase chain reaction showed strong induction of phaG, encoding the transacylase, under nitrogen starvation in P. putida KT2440 and the respective rpoN-negative mutant, indicating an RpoN-independent regulation of phaG. No transcription of phaG and no PHA accumulation was detected in the rpoN-negative mutant of P. aeruginosa neither from gluconate nor from octanoate as carbon source. Alginate-overproducing mutant P. aeruginosa FRD1 showed strongly decreased PHA accumulation from gluconate but no difference in phaC1 (encoding the PHA synthase) transcription, indicating that alginate biosynthesis competes with PHA biosynthesis regarding acetyl-CoA as precursor for both biopolymers. Transcription of phaF and phaI-F was nitrogen independent.

Acetyl Coenzyme A↗