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PubMed1995

Inhibitory action of metabolites of Pseudomonas aeruginosa against gram-negative bacteria.

Fifty clinical isolates of Pseudomonas aeruginosa were tested for inhibition of growth of clinical isolates of Escherichia coli, Salmonella infantis, Klebsiella pneumoniae and other Gram-negative bacteria in the authors' laboratory. Pseudomonas aeruginosa was strongly active against both E. coli and Enterobacter cloacae, with 89.4% and 94.7% inhibition respectively, but weakly active against S. infantis, K. pneumoniae and Proteus mirabilis with 56.3%, 48.8% and 23.8% inhibition, respectively. The pigmented strains were found to have stronger antimicrobial activity than the unpigmented strains. Pyocyanin, the major metabolite of Pseudomonas aeruginosa, has been shown to inhibit Escherichia coli, Proteus spp. and other Gram-negative bacteria, by research with a few strains of P. aeruginosa and a single inhibited strain. However, little attempt has been made to determine the inhibitory action of many strains of P. aeruginosa against a large number of clinical isolates such as Escherichia spp., Klebsiella spp., and Salmonella spp., up to now. For this reason, in this study we examined 50 randomly selected clinical isolates of P. aeruginosa for inhibition of growth of a wide range of Gram-negative bacteria, including 30 strains of E. coli, 30 of K. pneumoniae, 30 of S. infantis, 6 of Enterobacter cloacae and 9 of Proteus mirabilis.

Antibiosis↗
PubMed2004

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

Amino Acids↗
PubMed1978

Pattern of phenazine pigment production by a strain of Pseudomonas aeruginosa.

An atypical strain of Pseudomonas aeruginosa capable of synthesizing three phenazine pigments was isolated. Cultural conditions, under which the strain forms either chlororaphin, oxychlororaphin, or pyocyanine, are described. This broad spectrum of pigment production, as well as some other characteristics, sets this strain apart from previously described chlororaphin producers.

Ammonia↗
PubMed1994

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

Alkanes↗
PubMed2001

Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

Amino Acids↗
PubMed2001

Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when P. aeruginosa 57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when P. aeruginosa 57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.

Alginates↗
PubMed2002

Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa.

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.

Animals↗
PubMed1962

Evaluation of extracellular deoxyribonuclease activity in Pseudomonas.

Streitfeld, Murray M. (University of Miami, Coral Gables, Fla.), Edward M. Hoffmann, and Herbert M. Janklow. Evaluation of extracellular deoxyribonuclease activity in Pseudomonas. J. Bacteriol. 84:77-80. 1962.-Of 23 strains of P. aeruginosa isolated from pathological processes, 22 strains, representative of at least nine different somatic antigen types, were found to elaborate extracellular deoxyribonuclease. Qualitative determinations of enzyme elaboration were performed after the development of a convenient toluidine-blue assay plate procedure, dependent upon the metachromatic properties of the dye. This new procedure permits the determination of enzyme activity despite the presence, in the deoxyribonucleic acid-containing agar medium, of 2 to 5 ml/100 ml serum or plasma, or extracellular pyocyanin.

Culture Media↗
PubMed1962

Comparative taxonomy of crystallogenic strains of Pseudomonas aeruginosa and Pseudomon as chlororaphis.

Haynes, William C. (Northern Utilization Research and Development Division, Peoria, Ill.) and Lenora J. Rhodes. Comparative taxonomy of crystallogenic strains of Pseudomonas aeruginosa and Pseudomonas chlororaphis. J. Bacteriol. 84:1080-1084. 1962.-Only 11 of 39 strains received in the Agricultural Research Service Culture Collection under the designation Pseudonomas chlororaphis proved to be authentic; 28 were typical, pyocyanogenic strains of P. aeruginosa. The reason for this disproportionately high rate of misidentification apparently arises from an erroneous belief that the ability to produce green and yellow crystals of chlororaphin and oxychlororaphin is confined to P. chlororaphis. The ability of many strains of P. aeruginosa to do likewise is not well known. Inasmuch as the characteristic is not unique to P. chlororaphis, other criteria are required to distinguish crystallogenic strains of these species. After a taxonomic comparison of 18 strains of P. chlororaphis and 47 crystallogenic strains of P. aeruginosa, it was determined that there are three main distinctions: (i) P. aeruginosa grows well at 42 C but fails to grow upon serial transfer at 5 C, whereas P. chlororaphis fails to grow at 42 C, but grows well at 5 C: (ii) most strains of P. aeruginosa produce pyocyanin, whereas P. chlororaphis strains do not; (iii) P. aeruginosa cells possess only one or two polar flagella, whereas P. chlororaphis usually has at least four, sometimes as many as eight, polar flagella.

Flagella↗
PubMed1976

Fluorescent pseudomonads capable of growth at 41 degrees C but distinct from Pseudomonas aeruginosa.

One hundred and twenty-seven apyocyanogenic fluorescent Pseudomonas strains capable of growth at 41 degrees C, but differing from Pseudomonas aeruginosa, were typed serologically and tested for pyocin production, antibiotic susceptibility, selected biochemical reactions, and utilization of selected substrates. Results were compared with those from 40 apyocyanogenic and 14 pyocyanin-producing strains of P. aeruginosa. Unidentified fluorescent Pseudomonas (UFP) strains generally were not agglutinated by P. aeruginosa antisera and showed little or no pyocin activity. In contrast to P. aeruginosa strains, UFP strains usually failed to oxidize D-gluconate or reduce nitrate to nitrogen gas. They could not use D-gluconate or D-mannitol as sole carbon source and were susceptible to kanamycin. The cellular fatty acid compositions of major UFP groups resembled those of the alcaligenes-stutzeri groups.

Anti-Bacterial Agents↗
PubMed1982

Death of Pseudomonas aeruginosa in soil.

When incubated in natural (nonsterilized) soil, Pseudomonas aeruginosa died initially at a rate which approximated the rate for starvation of a pure culture in buffer. Predation by other soil microbes or phage did not appear to be involved, and pyocyanin either was not produced or was ineffective. The initial rate of death was followed by a second, considerably slower rate. Cells initially added in low numbers to soil also underwent biphasic death as above. Slow drying of the soil caused a period of rapid soil death of P. aeruginosa, but this then slowed to give residual numbers and a death rate similar to the second death rate noted for soil not allowed to dry. The cells in the dry soil had not changed genetically to a desiccation-resistant form. Pseudomonas aeruginosa died out completely in a relatively short time when the soil was first quickly dried to a water content similar to that obtained initially through slow drying and then further allowed to dry slowly. These observations appear to point to a dormant form, in some ways resembling a cyst, for P. aeruginosa in soil.

Humidity↗
PubMed1991

Effects of P. aeruginosa-derived bacterial products on tracheal ciliary function: role of O2 radicals.

The purpose of this investigation was to evaluate the effects of bacterial products derived from Pseudomonas aeruginosa on the function of airway cilia and to assess the role of phagocytes and oxygen radicals in the observed responses. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from normal sheep by brush biopsy (70% epithelial cells, 18% macrophages, 11% neutrophils). Baseline CBF ranged between 678 and 1,126 min-1. After 20 min of perfusion with the cell free supernatant of P. aeruginosa culture (mucoid strain), a concentration-dependent depression of CBF was observed with a 58% inhibition at a 1:1 dilution (P less than 0.05). The P. aeruginosa-derived products pyocyanin and 1-hydroxyphenazine also decreased CBF in a dose-related fashion. The cilion-inhibitory effects of the supernatant and bacterial products were markedly attenuated after centrifugation of the brush preparation (80% epithelial cells, 16.5% macrophages, 3.5% neutrophils). Glucose/glucose oxidase also caused a rapid, concentration-dependent cilioinhibition or ciliostasis. Catalase blocked or attenuated the ciliary effects of the supernatant, bacterial products and glucose/glucose oxidase. Thus bacterial products released from P. aeruginosa impaired ciliary activity by a pathway which involved neutrophils and was mediated by toxic oxygen radicals.

Animals↗
PubMed1994

Extracellular metabolites of Pseudomonas aeruginosa produce bronchoconstriction by different mechanisms.

Bacterial supernatants (BS) obtained from broth cultures of Pseudomonas aeruginosa cause bronchoconstriction in sheep, suggesting that BS contain proinflammatory metabolites. In this study we investigated the mechanism(s) responsible for this bronchial effect. BS were obtained from 48 h cultures and sterilized by filtration. Sheep (n = 6) were intubated and swallowed an esophageal balloon for the measurement of specific lung resistance (SRL). Aerosols of BS (3 ml total) immediately increased SRL (541%). Neither aerosolized broth (control) nor inhaled endotoxin in excess of that contained in the BS had an effect. BS challenges were repeated on separate occasions except that the sheep were treated 30 min before challenge with the anticholinergic agent atropine (0.2 mg/kg, intravenously); the anti-allergic agent nedocromil (1 mg/kg, aerosol); the histamine H1 antagonist chlorpheniramine (2 mg/kg); or the bradykinin (BK) B2 receptor antagonists NPC-567 (5 mg/ml, aerosol) or NPC-17761 (1 mg/ml aerosol). The results showed that greater than 90% protection (p < 0.05) was achieved when the animals were pretreated with atropine, nedocromil sodium, or either of the two BK antagonists, but only 27 +/- 21% protection was seen with chlorpheniramine pretreatment. These findings are characteristic of a BK-mediated response. Analysis of bronchoalveolar lavage fluid obtained before and after BS challenge confirmed that i-kinins, but not histamine, increased (p < 0.05) from 61 +/- 7 to 304 +/- 55 pg/ml. Control (broth) challenges produced no such change. To identify the metabolites involved, we tested the effects of aerosolizing two suspected components of BS, 1-hydroxyphenazine (1-HP) and pyocyanine (PYO) in five sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1992

Impairment of airway mucociliary transport by Pseudomonas aeruginosa products. Role of oxygen radicals.

We previously showed that the supernatant of a Pseudomonas aeruginosa (PA) culture and its constituents pyocyanin and 1-hydroxyphenazine inhibit ciliary activity of dispersed tracheal epithelial cells in vitro via the generation of oxygen radicals by phagocytes. In the present study, we wished to determine if tracheal mucus velocity (TMV) is also impaired by PA supernatant and if oxygen radicals have a mediating role. In conscious sheep, TMV (measured with a radiographic method) was determined before and serially following aerosol challenge with the cell-free supernatant of a PA culture or unconditioned culture medium (control). TMV decreased from a mean (+/- SEM) baseline of 6.7 +/- 1.1 mm/min (n = 6) by 29, 35, and 25% at 0.5, 3, and 24 h after challenge, respectively (p < 0.05), and returned to baseline 1 wk later (-6%, p = NS). Control medium had no effect on TMV (maximum decrease by 15% at 0.5 h). Aerosolized catalase blunted the effect of PA supernatant on TMV. To determine if the impairment of TMV involved ciliary inhibition, tissues were mounted in a chamber and ciliary beat frequency (CBF) and surface liquid velocity (SLV) were measured with a microscopic method. PA supernatant decreased both CBF (maximum mean decrease 12%; n = 5, p < 0.05) and SLV (maximum mean decrease 78%; n = 5, p < 0.05) in a dose-dependent fashion, with a correlation between the two parameters; these effects were blocked by catalase.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1991

Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium.

It has been suggested that proteinase enzymes could play an important role in the pathogenesis of chronic bronchial infections including bronchiectasis and cystic fibrosis (CF). Because Pseudomonas aeruginosa frequently colonizes the respiratory tract in bronchiectasis and CF, we examined the in vitro effects of human neutrophil elastase (HNE) and proteinase enzymes produced by P. aeruginosa (elastase: PE; alkaline proteinase: PAP) on the ciliary beat frequency (CBF) and ultrastructure of human nasal ciliated respiratory epithelium. HNE (500 micrograms/ml) progressively reduced CBF and caused marked epithelial disruption; lower concentrations (100 and 20 micrograms/ml) also caused epithelial disruption but without slowing CBF. The effects of HNE (500 micrograms/ml) were completely abolished by adding alpha 1-antitrypsin (5 mg/ml). There was no synergy between HNE and pyocyanin, a product of P. aeruginosa which slows CBF. PE in phosphate-buffered saline also caused epithelial disruption without slowing CBF; however, PE in medium containing divalent metal ions caused CBF slowing as well as epithelial disruption at 100 micrograms/ml. PAP (500 micrograms/ml) had almost no effect on ciliated epithelium. The effects of HNE and PE on nasal and bronchial epithelium obtained from the same patient were similar. Light and transmission electron microscopy revealed that HNE and PE were cytotoxic and caused detachment of epithelial cells from neighboring cells and the basement membrane. There was cytoplasmic blebbing of the cell surface and mitochondrial damage; however, no increase of abnormalities in the ultrastructure of cilia on living cells was seen. These results support the hypothesis that HNE and PE contribute to the delayed mucociliary clearance and epithelial damage that is observed in patients with chronic bronchial infection.

Bronchi↗
PubMed2005

Polyphosphate kinase 1 and the ocular virulence of Pseudomonas aeruginosa.

PURPOSE: To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa. METHODS: Using a mouse model of infection, P. aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence. These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment. RESULTS: The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P <0.016). Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea. However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P <0.006) than either the wild-type or restored strain. Furthermore, the PPK1-deficient mutant produced significantly less (P <0.022) pyocyanin. CONCLUSIONS: PPK1 is essential for a successful ocular infection by P. aeruginosa. The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response.

Animals↗
PubMed1979

Irgasan-induced pigmentation in Serratia marcescens and Pseudomonas aeruginosa.

Two irgasan-resistant micro-organisms (P. aeruginosa and S. marcescens) were used to study the effects of various antibiotic and chemotherapeutic agents on pigment production. These agents included streptomycin, thallium acetate, polymyxin B, hexachlorophene, irgasan, prodigiosin and DMSO (dimethyl sulphoxide). Only irgasan, compared to other drugs and membrane-active agents showed the unique property of inducing pigmentation in both P. aeruginosa and S. marcescens, i.e. prodigiosin in S. marcescens and pyocyanin in P. aeruginosa.

Dose-Response Relationship, Drug↗
PubMedDate not supplied

[Cultivation of Pseudomonas aeruginosa in media with chlorella fermentative hydrolysate as nutrient base].

The specific features of the growth of P. aeruginosa in media prepared with the use of Chlorella fermentative hydrolysate as nutrient basis were studied. The accumulation of microbial biomass was shown to depend on increased content of amino nitrogen in the media, while the effectiveness of the pyocyanin formation was linked with decreased content of amino nitrogen and a rise in the amount of sodium chloride.

Biomass↗