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Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.

Biodegradation, Environmental↗

Discontinuities in the evolution of Pseudomonas putida cat genes.

The organization and transcriptional control of chromosomal cat genes (required for dissimilation of catechol by the beta-ketoadipate pathway) in the Pseudomonas putida biotype strain (ATCC 12633) are reported. Nucleotide sequence reveals that catR is separated by 135 bp from the divergently transcribed catBC,A; catC begins 21 nucleotides downstream from catB, and catA begins 41 nucleotides downstream from catC. This contrasts with the gene arrangement in other bacteria, in which catA lies several kilobases upstream from catB. Properties of Tn5 mutants confirmed earlier suggestions that catR is a transcriptional activator and indicated that catA is activated by CatR independently of its activation of catBC. CatR binds to both a DNA fragment containing the catR-catB intergenic region and another DNA fragment containing catC. Pseudomonas strain RB1 resembles P. putida in some respects. Divergence of the two Pseudomonas chromosomes was revealed as nucleotide substitution of about 10% after alignment of known portions of catR,BC,A. Divergent transcriptional controls are suggested by a cluster of nucleotide sequence modifications in Pseudomonas strain RB1 which disrupt a stem-loop structure directly upstream of catB in the P. putida chromosome. Abrupt divergence of the catR,BC,A nucleotide sequences was achieved during evolution by insertion of an 85-bp palindromic genetic element uniquely positioned downstream from P. putida catR and counterpoised by insertion of a similar palindromic sequence in the Pseudomonas strain RB1 catB-catC intergenic region. Properties of the palindromic genetic element suggest that it may serve functions analogous to those of repetitive extragenic palindromic sequences and enteric repetitive intergenic consensus sequences in enteric bacteria.

Amino Acid Sequence↗

Pseudobactin biogenesis in the plant growth-promoting rhizobacterium Pseudomonas strain B10: identification and functional analysis of the L-ornithine N(5)-oxygenase (psbA) gene.

Pseudobactin(B10), the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligand D-N(5)-hydroxyornithine (D-N(5)-OH-Orn). We cloned the L-Orn N(5)-oxygenase (psbA) gene from a genomic library of Pseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of L-Orn to its N(5)-OH derivative. PsbA shows significant similarity to microbial omega-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The psbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor L-N(5)-OH-Orn, suggesting that L-Orn is hydroxylated before conversion to the D isomer. The L-Orn N(5)-hydroxylase-defective mutants of Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.

Iron↗

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Bacterial Proteins↗

Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp.

Phosphate starvation induced oligomeric proteins from the outer membranes of Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aureofaciens, and Pseudomonas chlororaphis were purified to homogeneity. The incorporation of the purified proteins into planar lipid bilayer membranes resulted in stepwise increases in membrane conductance. Single channel conductance experiments demonstrated that these proteins were all capable of forming small channels, similar to the Pseudomonas aeruginosa phospsate porin protein P, with average single channel conductances in 1 M KCl of between 233 and 252 pS. Single channel conductance measurements made in salts of varying cation or anion size indicated that the channels were uniformly anion selective. The measurement of single channel conductance as a function of KCl concentration revealed that all channels saturated at higher salt concentrations, consistent with the presence of an anion-binding site in the channel. Apparent Kd values for Cl- binding were calculated and shown to vary only twofold (180-297 mM) among all channels, including protein P channels. Phosphate competitively inhibited chloride conductance through these channels with apparent I50 values of between 0.59 and 2.5 mM phosphate at 40 mM Cl- and between 9.7 and 27 mM phosphate at 1 m Cl-. These data were consistent with the presence of a phosphate-binding site in the channels of these phosphate-regulated proteins. Furthermore, they indicated that these channels exhibit at least a 20- to 80-fold higher affinity for phosphate than for chloride.

Bacterial Outer Membrane Proteins↗

Degradation of acrylamide by immobilized cells of a Pseudomonas sp. and Xanthomonas maltophilia.

Two bacterial isolates capable of utilizing acrylamide as the sole source of carbon and nitrogen were isolated from herbicide-contaminated soil samples and identified as Pseudomonas sp. and Xanthomonas maltophilia. Batch cultures of Pseudomonas sp. and X. maltophilia completely degraded 62.8 mM acrylamide to acrylic acid and ammonia in 24 and 48 h, respectively. Pseudomonas sp. and X. maltophilia, when immobilized in calcium alginate, markedly increased the rate of degradation of acrylamide over batch cultures. Cells of the isolates immobilized in calcium alginate degraded acrylamide to acrylic acid and ammonia in less than 6 h. Initial metabolism of acrylamide by immobilized cells of Pseudomonas sp. followed by inoculation with nonimmobilized cells after 6 h totally removed acrylamide and its metabolites in 72 h. A similar procedure with X. maltophilia resulted in the total metabolism of acrylamide in 96 h. An inducible, intracellular amidase was responsible for the hydrolysis of acrylamide to acrylic acid and ammonia. The specific activity of Pseudomonas sp. amidase was higher than the specific activity of X. maltophilia amidase.

Acrylamide↗

Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8.

Respiratory epithelial cells play a crucial role in the inflammatory response during Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. In this study, we determined whether the binding of specific Pseudomonas gene products (pilin, flagellin) to their receptors on respiratory epithelial cells would result in production of the neutrophil chemoattractant IL-8. Piliated wild-type organisms, purified pili, or antibody to the pilin receptor (asialoGM1) evoked significant production of IL-8 by immortalized airway epithelial cells, whereas nonpiliated organisms were less able to bind to respiratory epithelial cells and stimulated much less IL-8 secretion (P < 0.01). A piliated, nonflagellated strain was also associated with decreased binding and a diminished level of IL-8 production when compared to wild-type organisms. Isogenic, nonadherent rpoN mutants, lacking pilin and flagellin, did not bind or elicit an IL-8 response. In addition, the IL-8 response was four-fold higher in a cystic fibrosis cell line compared with its corrected cell line. The Pseudomonas autoinducer, an exoproduct secreted during chronic infection, was found to stimulate IL-8 in a dose-dependent manner. P. aeruginosa adhesins, which are necessary for initial infection, directly stimulate IL-8 production by respiratory epithelial cells and therefore play a major role in the pathogenesis of Pseudomonas infection in patients with cystic fibrosis. The inflammatory response is subsequently perpetuated by Pseudomonas autoinducer which is secreted during chronic infection.

Adhesins, Bacterial↗

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Base Pair Mismatch↗

Active lactonizing lipase (LipL) efficiently overproduced by Pseudomonas strains as heterologous expression hosts.

Pseudomonas sp. strain 109 secretes lactonizing lipase (LipL), which catalyzes efficient intramolecular transesterification of omega-hydroxyfatty acid esters to form macrocyclic lactones. Because Escherichia coli was found to be unsuitable as an expression host due to the predominant formation of inactive LipL-inclusion bodies and a lack of proper secretion machinery which is also required for the formation of active LipL, Pseudomonas strains were surveyed as expression hosts. Pseudomonas sp. strain 109, an original LipL producer, showed a 7.1-fold higher level of active LipL when the lipL gene under the control of tac-lacUV5 tandem promoter was introduced together with a limL gene encoding a LipL-specific chaperon. Pseudomonas aeruginosa ADD 1976 containing a T7 RNA polymerase gene in the chromosome and plasmid-borne lipL-limL genes under the control of T7 promoter showed a 13-fold higher level of active LipL. Several combinations in the number of lipL and/or limL genes on the plasmid were investigated, and (lipL)3-limL was found to be most efficient, yielding a 67-fold greater production of active LipL than that obtained by the wild-type Pseudomonas sp. strain 109.

Bacterial Proteins↗

Cadexomer iodine in ulcers colonised by Pseudomonas aeruginosa.

The clinical and bacteriological efficacy of topical cadexomer iodine on venous leg ulcers colonised with Pseudomonas aeruginosa was evaluated in an open, uncontrolled, multicentre pilot study. Nineteen patients with venous leg ulcers (median ulcer area 15.5 cm2) were treated with cadexomer iodine paste and short-stretch bandaging for 12 weeks or until the ulcer was healed within that period. Bacteriological cultures for growth of Pseudomonas aeruginosa and ulcer area measurements were regularly performed during the study period. Seven patients were withdrawn during the study period for the following reasons: pain in the ulcer (n = 2), ulcer enlargement (n = 2), treatment of cellulitis in non-study ulcer with a systemic antibiotic that is effective against Pseudomonas aeruginosa (n = 1), increased thyroid-stimulating hormone level (n = 1) and poor compliance (n = 1). In addition, one patient was erroneously withdrawn. After one week's treatment, 11 out of 17 patients (65%) had a negative culture of Pseudomonas aeruginosa. At 12 weeks, six out of eight patients (75%) had a negative culture and complete healing was achieved in three patients (16%). The median ulcer area reduction obtained at 12 weeks was 32.9%. The results indicate that cadexomer iodine paste might be the treatment of choice for venous leg ulcers colonised with Pseudomonas aeruginosa. However, a larger controlled clinical study is needed for final evaluation.

Adult↗

The metabolism of caffeine by a Pseudomonas putida strain.

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.

Caffeine↗

Degradation of lawsone by Pseudomonas putida L2.

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.

Biotransformation↗

[In vitro adherence of Staphylococcus epidermidis and Pseudomonas alcaligenes to intraocular lenses].

PURPOSE: To quantify and compare the in vitro adherence of Staphylococcus epidermidis and Pseudomonas alcaligenes to different intraocular lenses (IOLs). METHODS: Fourteen intraocular lenses were used in the experiment. Four of polymethylmethacrylate (PMMA), four of silicone, four of hydrogel and two of acrylic. Eight intraocular lenses were placed in eight test tubes containing 4 ml of Pseudomonas alcaligenes suspension, and six intraocular lenses were placed in six test tubes containing 4 ml of Staphylococcus epidermidis suspension. The bacterial suspension used for adherence tests was 10(8) colony-forming units per milliliter (CFU/mL) which corresponds to 0.5 in the scale of McFarland. The lenses were incubated at 37 degrees for two hours. After this, intraocular lenses were removed from the test tubes and dried twice with the use of distilled and sterile water. The material was spread on blood-agar for cultivation at 35-37 degrees C and was evaluated every 24 hours up to 72 hours. In the samples where there was bacterial growth, the colonies were counted using the conventional laboratory methods. All assays were performed in duplicate. RESULTS: Adherence of Staphylococcus epidermidis to PMMA intraocular lenses was lower than to hydrogel and silicone intraocular lenses. Adherence of Pseudomonas alcaligenes to hydrogel intraocular lenses was lower than to PMMA, acrylic and silicone intraocular lenses. CONCLUSIONS: Results suggest that in vitro adherence of Staphylococcus epidermidis and Pseudomonas alcaligenes to intraocular lenses is influenced by type of material of the lens and by microorganism species. Bacterial adherence may play a role in the pathogenesis of some forms of endophthalmitis after cataract surgery. More in vitro and clinical studies are necessary to elucidate the mechanisms by which Staphylococcus epidermidis and Pseudomonas alcaligenes cause endophthalmitis.

Bacterial Adhesion↗

Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk.

In the present study, we investigated the effect of Pseudomonas spp. growth on the plasmin enzymatic system in casein and whey fractions of fresh milk. Two bacterial strains, Pseudomonas spp. SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into fresh milk and incubated at 7 degrees C for 3 d. Bacterial counts were approximately 10(8) cfu/ml by d 3. Samples collected every 24 h were treated to separate the casein from the whey fraction. Casein and whey fractions were subjected to electrophoresis to visualize protein breakdown and plasmin activity and to colorimetric assays to quantify plasmin-related activities. With psychrotrophic bacterial growth, plasmin levels in casein fractions decreased significantly and in whey fractions increased then decreased significantly. Fresh milk results were similar for the two strains and were similar to earlier results with reconstituted nonfat dry milk. A transmission electron microscopy study by immunocytochemistry showed the presence of plasmin in casein micelles and its disappearance upon microbial growth in the milk. We hypothesized that extracellular microbial proteases produced by psychrotrophic microorganisms are responsible for this effect. To confirm this, an extracellular bacterial protease was isolated from Pseudomonas fluorescens M3/6 by ammonium sulfate fractionation and ion-exchange chromatography and incubated with fresh milk. Milk samples analyzed during incubation with the protease had significantly increased plasmin and plasminogen activities in the whey fraction within 5 h of incubation, while differences in activities in the casein fraction occurred at time 7.5 h for plasmin activity and 10 h of incubation for plasminogen activity. These quantitative data were supported by plasmin activity as visualized by casein-SDS-PAGE. These results suggest that growth of the Pseudomonas strains in fresh milk, and particularly their production of extracellular proteases, may be a causative factor in the release of plasmin from the casein micelle. Such plasmin release could affect the quality of cheeses and other food products that utilize dairy ingredients.

Animals↗

[Effect of macrolides on biofilm of mucoid Pseudomonas aeruginosa].

OBJECTIVE: To observe the effect of macrolides with different structures on biofilm of mucoid Pseudomonas aeruginosa so as to provide a theoretical basis for treatment of bacterial biofilm diseases. METHODS: Modified plate culture method was used to establish in vitro bacterial biofilm model, which was identified by silver nitrate staining. After the biofilm was treated by gatifloxacin and/or macrolides with different structures and of different concentrations, the number of viable bacteria was measured by MTT method and bacterial adherence was measured by crystal violet staining. RESULTS: Clarithromycin and azithromycin of 1/4 and 1/16 MIC combined with gatifloxacin significantly inhibited the adherence of mucoid Pseudomonas aeruginosa (P < 0.05) and reduced the number of viable mucoid Pseudomonas aeruginosa compared with gatifloxacin alone (P < 0.05), while such effects could not be seen for rovamycin. CONCLUSION: Inhibiting the adherence of mucoid Pseudomonas aeruginosa may be one of the mechanisms by which macrolides such as clarithromycin and azithromycin enhance the antibacterial activity of gatifloxacin against mucoid Pseudomonas aeruginosa.

Anti-Bacterial Agents↗

[The assay of beta-lactamase activity in Pseudomonas aeruginosa biofilm].

OBJECTIVE: To analyze the resistance mechanism of Pseudomonas aeruginosa biofilm bacteria. METHODS: An in vitro model of Pseudomonas aeruginosa bacterial biofilm was established in silicon disk with modified flat-board method and a rapid staining procedure of AgNO3 was used to verify it. Biofilm was observed under scanning electron microscopy. The test was carried out in three groups. Group A was planktonic bacteria of Pseudomonas aeruginosa, group B biofilm bacteria of Pseudomonas aeruginosa and group C biofilm bacteria induced by imipenem. The activity of beta-lactamase was quantitated with a spectrophotometric assay method and beta-lactamase quantitation was determined by using Bio-Rad protein assay. RESULTS: The activity and protein content of beta-lactamase in group B was higher than that in group A by 4.67 and 2.09 times. The activity of group C was 21.86 times as much as that in group A and 4.68 times as much as that in group B. The quantitation in group C was 6.28 times as much as that in group A and 3.00 times as much as that in group B. The activity and quantitation of the three groups were different significantly from each other (P < 0.01). CONCLUSION: The production of beta-lactamase in Pseudomonas aeruginosa biofilm bacteria is one of the main reasons of its resistance.

Anti-Bacterial Agents↗

Cadmium and zinc sensitivity and tolerance in Bacillus subtilis subsp. niger and in a Pseudomonas sp.

The action of Cd2+ and Zn2+ on Bacillus subtilis subsp. niger ATCC 9372, and on a Pseudomonas sp. (possibly Pseudomonas fluorescens), isolated from cadmium-polluted soil has been determined and compared with results obtained previously with Klebsiella aerogenes. In liquid medium the lag and the mean generation time of Bacillus subtilis subsp, niger increased with increasing Cd2+ or Zn2+ concentrations whereas only the total biomass of the Pseudomonas sp. was affected. Nevertheless, the responses of both species indicated a specific action at low concentrations and a more general toxic action at high concentrations. The survival on Cd2+ - or Zn2+ - agar depended on the state of the metal ions with regard to chelating ligands and on the nutritional stage of the organisms. In admixture, the metal ions acted synergistically, particularly on the Pseudomonas sp. Resistance to both metal ions developed. It was graded to the training concentration and reciprocal cross-resistance occurred with Bacillus subtilis. subsp. niger but not with the Pseudomonas sp.

Bacillus subtilis↗

Comparative study of the histologic reactions to intravenous injections of heat-killed Pseudomonas aeruginosa and of BCG.

Intravenous injection (i.v.) of heat-killed Pseudomonas aeruginosa in mice produced histologic changes in the thymic cortex, some of which resembled, while others differed from, those produced by i.v. injection of living BCG. The changes that were similar consisted of pyroninophilia of cortical lymphocytes and hyperplasia of epithelial cells in the medulla and at the corticomedullary junction with increased PAS positive cells and secretions. Major differences, however, in the sequence and nature of the histologic events were observed. Pseudomonas injections produced thymic epithelial cell hyperplasia with increased PAS positive cells and secretions and pyroninophilia of thymic cortical lymphocytes earlier than did i.v. BCG (day 1 versus day 7). Corticomedullary inversion of thymic structure and early transient hyperplasia of the thymus dependent areas in the lymph nodes and spleen occurred after Pseudomonas but not after BCG injections. Hyperplasia in the B cell areas and germinal centers started to appear at day 10 after injection of Pseudomonas and persisted up to day 21 (compared to day 7 and day 14, respectively, for BCG). In contrast to i.v. BCG, Pseudomonas injections did not produce granulomas or macrophage proliferations.

Animals↗