Toxic cyanobacteria in the Netherlands

Summary In the summer of 1980 waterblooms and scums ofMicrocystis aeruginosa, Oscillatoria agardhii andGloeotrichia echinulata have been collected from 11 locations. Acute toxicity of sonificated algal suspensions was tested by intraperitoneal injection in mice. The results indicate that in 9 out of 10 sample sites lethalM.aeruginosa hepatoxin(s) were present, while in most samples associated bacterial toxin(s) were possibly involved in SDF (Slow Death Factor) intoxication symptoms and death. Two unialgal strains ofM.aeruginosa (RID-2B, isolated from a Dutch storage reservoir, and the Norwegian toxic CYA 57) showed the same toxicity.Suspensions ofO.agardhii were also found to be lethal. Symptoms and mortality are thought to be attributed to at least two toxic factors. One resembling FDF, the other possibly resulting from associated bacteria. Administration ofG.echinulata suspensions did not kill mice but produced symptoms of illness, which were not consistent with those observed withMicrocystis FDF (Fast Death Factor) or SDF.Because there are no documented cases of health impairments due to cyanobacteria in The Netherlands, an inquiry was held in autumn 1980 and 1981 among 280 physicans practising in Rijnmond, into the occurrence of primary or allergic dermatitis and conjunctivitis in humans after swimming in waterblooms of toxicM.aeruginosa in Lake Brielle. In this inquiry only a few cases of skin reactions and conjunctivitis were reported.     

Comparative study of immunobiological activities ofPseudomonas aeruginosa andBrucella melitensis lipopolysaccharides

The toxic activity ofBrucella melitensis andPseudomonas aeruginosa lipopolysaccharides as well as their behavior as immunogens, mitogens, and interferon inducers have been studied. Although their toxicities were very similar, the former molecule was incapable of eliciting a primary immune response in mice. Rabbit hyperimmunization gave titers half of those obtained withP. aeruginosa lipopolysaccharide. Optimal mitogenic responses of spleen cell cultures were obtained using 10–50 μg/ml and 50–100 μg/ml ofPseudomonas andBrucella lipopolysaccharide, respectively, giving the latter a lower stimulation of³H-thymidine uptake. Interferon titers induced in chickens byBrucella lipopolysaccharide were three times lower than those obtained withPseudomonas lipopolysaccharide.     

Recombinant outer membrane protein F ofPseudomonas aeruginosa elicits antibodies that mediate opsonophagocytic killing,but not complement-mediated bacteriolysis,of various strains ofP. aeruginosa

Recombinant outer membrane protein F ofPseudomonas aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Rats were immunized intramuscularly with 25 g of recombinant protein F adsorbed to aluminum hydroxide adjuvant on days 1, 14, and 28 and then challenged on day 42 via intratracheal inoculation of agar beads containing cells of a clinical isolate ofP. aeruginosa. On day 49 the lungs were examined macroscopically for the presence and severity of lesions and submitted for quantitation of the bacteria present. The recombinant protein F vaccine afforded significant protection against subsequent challenge withP. aeruginosa in the immunized rats, as compared with control rats immunized with bovine serum albumin. Antisera from the recombinant protein F-immunized rats mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells ofP. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant ofP. aeruginosa. The antisera to recombinant protein F did not promote complement-mediated bacteriolysis ofP. aeruginosa. These data demonstrate that recombinantP. aeruginosa protein F has efficacy as a protective vaccine in a rat model of chronic pulmonary infection.     

Gentamicin-resistantPseudomonas aeruginosa: Concepts regarding their evolution and attenuated virulence

Pseudomonas aeruginosa, a free-living bacterial species, is a major nosocomial pathogen, especially of compromised patients within medical facilities. Numerous factors contribute to the ecological selection of this bacterial species within the hospital environment, among which the expression of newly acquired or quiescent enzymatic capability seems par-amount. The emergence of pathogenic strains ofP. aeruginosa appears to be gradual, embodying a transition of strains from their natural aquatic environment, to establishing inanimate (hospital) and animate (human) reservoirs. In this stepwise transition, subsets ofP. aeruginosa may evolve which express a survival trait, for example, gentamicin resistance, but concomitantly suffer a loss of invasive potential. In this study,P. aeruginosa strains from natural [22], hospital [11], and stool [17] sources were evaluated for their physiological and exoenzymatic activity and compared with gentamicin-resistantP. aeruginosa (GRPA) strains [49] of clinical origin. As a whole, environmental and hospital isolates showed reduced enzymatic potential, for example, frequency of production of elastase, lipase, deoxyribonuclease, and pyocyanin production. Human fecal isolates most closely resembled the prototype of human invasiveP. aeruginosa in their gentamicin susceptibility (95%) and increased frequencies of exoenzymes, including elastase production. On the other hand, GRPA were frequently apyocyanogenic (9/49), lacked extracellular enzymes correlated with pathogenicity, and were rarely isolated from systemic sites. When encountered, these strains appeared to represent colonization of a body site rather than incitants of overt infection. As a subset ofP. aeruginosa, gentamicin resistance was seen predominantly among serotype 11 strains, and encountered most frequently from patients with localized urinary tract infections.     

Biological control of root rot-root knot disease complex of tomato

Efficacy of Pseudomonas aeruginosa alone or in combination with Paecilomyces lilacinus was evaluated in the control of root-knot nematode and root-infecting fungi under laboratory and field conditions. Ethyl acetate extract (1 mg/ml) of P. lilacinus and P. aeruginosa,respectively, caused 100 and 64% mortality of Meloidogyne javanica larvae after 24 h. Ethyl acetate fractions of biocontrol agents were more effective than hexane extracts in the suppression of M. javanica larvae, indicating that active nematicidal compounds are intermediary in polarity. In field experiments, biocontrol fungus and bacterium significantly suppressed soilborne root-infecting fungi including Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Meloidogyne javanica, the root-knot nematode. P. lilacinus parasitized eggs and female of M. javanica and this parasitism was not significantly influenced in the presence of P. aeruginosa. P. aeruginosa was reisolated from the inner root tissues of tomato, whereas P. lilacinusdid not colonize tomato roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fungi associated with sorghum seeds

Summary A study of the external and internal fungi associated with different varieties ofSorghum seeds has been made. The varieties tested included eighteen local varieties and twelve newSorghum varieties obtained from the Rockefeller Foundation. The external fungi were studied by preparing suspensions of superficial fungi and growing on potato dextrose agar. The internal fungi were studied by planting surface sterilized seeds on P.D.A. and pure cultures of all these fungi were prepared.The external fungi found to be associated with the different varieties included different species ofPhycomyces, Circinella, Syncephalastrum, Chaetomium, Curvularia, Cladosporium, Helminthosporium, Montospora, Pullularia, Aspergillus, Penicillium, Cephalosporium, Trichoderma, Phoma, Fusarium.The internal fungi recovered from these varieties included species ofChaetomium, Cladosporium, Curvularia, Helminthosporium, Heterosporium, Hormodendron, Pullularia, Alternaria, Aspergillus, Blastomyces, Monilia, Penicillium, Fusarium, Phoma, Phomopsis. Varieties 4403B, 1060 and 503 were found to carry a large number of fungi. Variety Black spanish was found to be entirely free from any internal or external fungus. Varieties which were free from endophytic fungi but possess external fungi only were Kaoling 301, African variety 901 and Shallus 475. Three fungi viz.,Pullularia, Heterosporium, Monilia have been recovered from theSorghum seeds for the first time.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Biophysical characterization of OprB,a glucose-inducible porin ofPseudomonas aeruginosa

OprB, a glucose-inducible porin ofP. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with aK _s for glucose of 380 ± 40 mM, and the formation of channels with a strong selection for anions. Analysis ofP. aeruginosa OprB circular dichroism spectra revealed a high sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB ofP. putida [Saravolacet al. (1991).J. Bacteriol. 173, 4970–4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. WhereasP. aeruginosa OprB is anion selective,P. putida OprB and other carbohydrate selective porins are known to be cation selective.     

Aerobic respiratory activity of the yeastlike phase of Sporotrichum schenckii as affected by high hydrogen ion concentrations

Summary Six strains ofSporotrichum schenckii were studied in regard to the effect of hydrogen ion concentration on certain aspects of the aerobic respiratory activity of the yeastlike phase.Optimal oxygen uptake of endogenous respiration occurred at pH 2.0, although no effects were observed on the oxygen-carbon dioxide exchange ratio (respiratory quotient) at this pH value when compared to the R.Q. obtained at pH 7.0. Endogenous respiratory activity at both pH 2 and 7 was markedly sensitive to the presence of certain respiratory inhibitors.Optimal respiratory activity using glucose as substrate occurred at pH 7.0 .On the other hand, oxidation of pyruvate as substrate proceeded at significant rates only at pH values below pH 4.0. With decreasing hydrogen ion concentration, accumulation of this organic acid occurred when glucose was employed as substrate. With the exception of acetate, none of the organic acid respiratory intermediates were found to stimulate respiration.The results reported herein suggest that the respiratory activity of the yeastlike phase ofS. schenckii differs in several respects from that observed for the yeastlike phases ofHistoplasma capsulatum andBlastomyces dermatitidis.From the Research Laboratory, Veterans Administration Hospital, Kansas City, Missouri, and The Department of Microbiology, Kansas University Medical Center, Kansas City, Kansas.Supported in part by USPHS Grant AI 03485.     

Chloramphenicol acetyltransferase fromPseudomonas aeruginosa—a new variant of the enzyme

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100–1,000 g/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are highly resistant to the drug. In an attempt to understand the resistance mechanism ofP. aeruginosa to chloramphenicol, CAT produced by this microorganism was examined and compared with other known variants of the enzyme which are usually associated with high-level resistance in other species. CAT ofP. aeruginosa was found to be synthesized constitutively and to have a molecular weight of 22,500 per protomer. The specific activity of the enzyme is 30-fold lower than that ofEscherichia coli type II CAT. The same specific activity was obtained for CAT produced by two isolates ofP. aeruginosa, one with high and one with low resistance. The elution patterns from affinity and hydrophobic chromatography,K _m 's for chloramphenicol, the high affinity of the enzyme for acetyl-CoA (2- to 6-fold greater than that of any other variant) and insensitivity to sulfhydryl reagents indicate that the properties of this enzyme are not identical with those of any of the known variants.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Autolytic nature of iridescent lysis inPseudomonas aeruginosa

Attempts to demonstrate a filterable agent to be the cause of iridescent lysis inPseudomonas aeruginosa were uniformly negative. It was not possible to transmit the principle by needle transfer from iridescent plaques to non-iridescent cultures, and plaques produced byP. aeruginosa bacteriophages were never iridescent. Iridescent lysis and bacteriophage lysis were subjected to antibiotics, anti-metabolites, agar at different pH values, antisera to bacteriophage-lysed and to iridescent-lysed bacteria, different oxygen concentrations, and to different nutritional sources. Certain antibiotics, notably tetracycline, streptomycin and polymyxin inducedde novo or enhanced formation of metallic lysis in nutrient agar surface cultures ofP. aeruginosa. Bacteriophage was not induced. Antimetabolites of amino acids, carbohydrates and vitamins inhibited iridescence, or inhibited it at high concentrations and enhanced it at low concentrations. Bacteriophage action was unaffected. Metallic lysis was completely inhibited at pH 6.0; it was inhibited on media containing dye or bile salt and at lowered oxygen concentrations. Bacteriophage action was not affected under these conditions. Antisera to iridescent lysates and to bacteriophage lysates ofP. aeruginosa were tested. Phage antiserum strongly neutralized phage lysis but had no effect on iridescent lysis; antisera to iridescent lysates had no effect on either. No evidence for phage mediation of iridescent lysis was seen in any of the experiments. Iridescent lysis ofP. aeruginosa was demonstrated to be based on metabolic autolysis.     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.     

Pseudomonas aeruginosa binds to soluble cellular fibronectin

We have investigatedPseudomonas aeruginosa binding to plasma and cellular fibronectin (FN), in both their soluble and insoluble forms. Bacterial binding to insoluble FN was studied by exposing coverslips coated with FN to radiolabeled microorganisms.P. aeruginosa binding to soluble FN was investigated (1) by comparing radiolabeled bacteria treated with FN with PBS-treated bacteria in their adhesion to a collagen matrix; (2) by analyzing the reactivity ofP. aeruginosa with plasma or cellular FN adsorbed to gold particles with transmission electron microscopy (TEM).P. aeruginosa did not bind significantly to insoluble plasma or cellular FN, or to soluble plasma FN. In contrast, bacterial treatment with soluble cellular FN significantly increased the adhesion to the collagen matrix. With TEM, we confirmed the reactivity ofP. aeruginosa with soluble cellular FN. Because there is a marked secretion of cellular FN during wound repair, we speculate that this reactivity may account for the propensity ofP. aeruginosa to infect repairing tissues.     

Phagocytic inhibitory activity in serum of cats immunized withPseudomonas aeruginosa lipopolysaccharide

Sequentially collected sera from cats chronically immunized withPseudomonas aeruginosa lipopolysaccharide (LPS) serotype 5 were assessed for their effect on phagocytosis by alveolar macrophages from nonimmunized cats. Phagocytosis was measured by incubating macrophage monolayers for 20 min in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Sustained phagocytic inhibitory activity developed in the sera of eight of 11 immunized cats (mean inhibition ranged from 30% to 73%) after 13 weekly immunizations. The activity was specific because phagocytosis ofStaphylococcus aureus andP. aeruginosa of another serotype (type 6) was unimpaired. An enzyme-linked immunosorbent assay showed an increase in the amount of LPS-specific IgG in postimmunization sera from the eight cats with inhibitory activity. The IgG appeared to be serotype specific because significantly higher titers were obtained against LPS serotype 5 than LPS serotype 6. The results suggest that phagocytic inhibitory activity in sera from LPS-immunized cats may be due to anti-LPS IgG.     

Soil bacteria parasitic on Azotobacteriaceae

Deliberate enrichment of garden soil led to the isolation of anAchromobacter ¹ capable of lysing various Azotobacteriaceae on agar plates. Lysis was less pronounced in liquid media; attempts to demonstrate diffusible lytic factors were not successful. Thirty-four other microbes, freshly isolated from soils or obtained from collections, had no activity; two strains out of seven ofPseudomonas aeruginosa showed some degree of lytic action.     

Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 10⁷ colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> phage endolysin can enhance permeability of<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> outer membrane and induce cell lysis

To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic -lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.