Analysis of the draft genome of Pseudomonas fluorescens ATCC17400 indicates a capacity to take up iron from a wide range of sources,including different exogenous pyoverdines

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.     

Resistance of biofilms containing alginate‐producing bacteria to disintegration by an alginate degrading enzyme (Algl)

Pure culture biofilms of Pseudomonas aeruginosa (strains 8830 and ATCC 700829) and mixed population biofilms composed of Pseudomonas aeruginosa (ATCC 700829), Pseudomonas fluorescens (ATCC 700830), and Klebsiellapneumoniae (ATCC 700831) were treated with an alginate‐degrading enzyme (AlgL). The enzyme effectively depolymerized the mannuronic acid rich (92%), partially O‐acetylated bacterial alginate produced by P. aeruginosa (8830), both in dilute solution and in a gel‐like, concentrated state. However, both biofilms were unaffected by the presence of the enzyme. These findings suggest either that bacterial alginates do not contribute significantly to the cohesiveness of biofilms or that the alginate is protected from enzymatic degradation in biofilms.     

Isolation of a Pseudomonas sp. Which Utilizes the Phosphonate Herbicide Glyphosate

A strain of bacteria has been isolated which rapidly and efficiently utilizes the herbicide glyphosate (N-phosphonomethylglycine) as its sole phosphorus source in a synthetic medium. The strain (PG2982) was isolated by subculturing Pseudomonas aeruginosa ATCC 9027 in a synthetic broth medium containing glyphosate as the sole phosphorus source. Strain PG2982 differs from the culture of P. aeruginosa in that it is nonflagellated, does not produce pyocyanin, and has an absolute requirement for thiamine. Strain PG2982 has been tentatively identified as a Pseudomonas sp. strain by its biochemical activities and moles percent guanine plus cytosine. Measurements of glyphosate with an amino acid analyzer show that glyphosate rapidly disappears from the medium during exponential growth of strain PG2982. In batch culture at 30°C, this isolate completely utilized 1.0 mM glyphosate in 96 h and yielded a cell density equal to that obtained with 1.0 mM phosphate as the phosphorus source. However, a longer lag phase and greater generation time were noted in the glyphosate-containing medium. Strain PG2982 can efficiently utilize glyphosate as an alternate phosphorus source.     

Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.     

Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level

The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis.     

Protective Effect of DNA Vaccine Encoding Pseudomonas Exotoxin A and PcrV against Acute Pulmonary P. aeruginosa Infection

Infections with Pseudomonas aeruginosa have been a long-standing challenge for clinical therapy because of complex pathogenesis and resistance to antibiotics, thus attaching importance to explore effective vaccines for prevention and treatment. In the present study, we constructed a novel DNA vaccine by inserting mutated gene toxA_m encoding Pseudomonas Exotoxin A and gene pcrV encoding tip protein of the type III secretion system into respective sites of a eukaryotic plasmid pIRES, named pIRES-toxA_m-pcrV, and next evaluated the efficacy of the vaccine in murine acute Pseudomonas pneumonia models. Compared to DNA vaccines encoding single antigen, mice vaccinated with pIRES-toxA_m-pcrV elicited higher levels of antigen-specific serum immunoglobulin G (IgG), enhanced splenic cell proliferation and cytokine secretion in response to Pseudomonas aeruginosa antigens, additionally PAO1 challenge in mice airway resulted in reduced bacteria burden and milder pathologic changes in lungs. Besides, it was observed that immunogenicity and protection could be promoted by the CpG ODN 1826 adjuvant. Taken together, it’s revealed that recombinant DNA vaccine pIRES-toxA_m-pcrV was a potential candidate for immunotherapy of Pseudomonas aeruginosa infection and the CpG ODN 1826 a potent stimulatory adjuvant for DNA vaccination.     

All-D-cecropin B: Synthesis,conformation, lipopolysaccharide binding,and antibacterial activity

Cecropin B (LCB) is a natural peptide with antibacterial and antifungal properties. The enantiomer of LCB, containing all-D amino acids (DCB), was synthesized to examine its antibacterial and binding properties. The conformation of DCB was compared to its enantiomer by circular dichroism. Both the L- and D-peptides showed an identical induction of -helical secondary structure. However, binding studies between Lipopolysaccharide (LPS) and DCB or LCB were studied with a dimethylmethylene blue spectrophotometric assay, showing the two enantiomeric peptides differed in their interaction with LPS. Antibacterial activity of DCB was determined against three Gram-negative bacteria, Pantoea agglomerans (ATCC 27996), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 17648), giving comparable results to LCB.     

Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

Unsaturated Fatty Acids Promote the Phagocytosis of P. aeruginosa and R. equi by RAW264.7 Macrophages

In the present study, using the murine monocyte/macrophage cell line RAW264.7 as a model system, we analyzed the phagocytosis rate and the bactericidal capacity of polyunsaturated fatty acids (PUFA)-enriched macrophages against Pseudomonas aeruginosa and Rhodococcus equi. The P. aeruginosa strain ATCC 10145, the virulent R. equi strain ATCC 33701, and the non-virulent R. equi strain ATCC 6939 were examined. Flow cytometric detection of intracellular microorganisms in combination with viability assays were used to determine the impact of PUFA on the number of engulfed, surviving as well as replicating bacteria. Macrophage enrichment with PUFA resulted in an increase of the internalization rate of the microorganisms by the immune cells. Moreover, an impeding action of the unsaturated fatty acids on the intracellular survival rates of the virulent strains P. aeruginosa ATCC 10145 and R. equi ATCC 33701 could be observed. The n-3 fatty acid docosahexaenoic acid (DHA) as well as the n-6 fatty acid arachidonic acid (AA) showed the most pronounced effects. Taken together, our data support the idea of supplementing PUFA to immunocompromised individuals as well as to people suffering from chronic infections with P. aeruginosa or R. equi to improve macrophage phagocytic and microbicidal activity.     

Clinical case: Combined pulmonary fibrosis and emphysema with pulmonary hypertension – clinical management

Background

Extended spectrum ß-lactamases (ESBLs) represent a major group of lactamases responsible for resistance, mostly produced by gram-negative bacteria, to newer generations of ß-lactam drugs currently being identified in large numbers worldwide. The present study was undertaken to see the frequency of ESBL producing Pseudomonas spp. isolated from six hundred clinical specimens (wound, pus, aural, urine, sputum, throat and other swabs) collected over a period of three years from two tertiary care hospitals in Bangladesh.

Findings

Aerobic bacterial culture was performed on aseptically collected swabs and only growth of Pseudomonas was considered for further species identification and ESBL production along with serotyping of Pseudomonas aeruginosa. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer agar diffusion method and ESBL production was detected on Mueller Hinton agar by double-disk synergy technique using Amoxicillin-Clavulanic acid with Ceftazidime, Cefotaxime, Ceftriaxone and Aztreonam. Culture yielded 120 Pseudomonas spp. and 82 of them were biochemically characterized for species. Pseudomonas aeruginosa was found to be the predominant (90.2%) species. Of 82 isolates tested for ESBL, 31 (37.8%) were ESBL positive with 29 (93.5%) as Pseudomonas aeruginosa, the remaining 2 (6.5%) were Stenotrophomonas maltophilia and Ralstonia pickettii. Antibiogram revealed Imipenem as the most effective drug (93.3%) among all antimicrobials used against Pseudomonas spp. followed by Aminoglycosides (63.7%).

Conclusion

ESBL producing Pseudomonas spp. was found to be a frequent isolate from two tertiary care hospitals in Bangladesh, showing limited susceptibility to antimicrobials and decreased susceptibility to Imipenem in particular, which is a matter of great concern.     

Cytochrome c-556, a di-heme protein from Pseudomonas aeruginosa

A procedure is described for the purification of cytochrome c-556 from Pseudomonas aeruginosa. The isolated hemoprotein exists as a dimer with a molecular weight of approximately 77 200. The dimer can be dissociated into a monomeric species (or single polypeptide chain) of 40 500 molecular weight by means of sodium dodecyl sulfate or 4 M urea. The amino acid composition demonstrates the presence of four half-cystine residues per 43 000 molecular weight. Heme and iron analyses indicate that two c-type hemes are covalently linked to each polypeptide chain. The absorption spectrum of ferrocytochrome c-556 has a double α-band with a peak at 556 nm and a shoulder at 552 nm; the β-band appears at 521 nm and the Soret band at 420 nm.The electron paramagnetic resonance spectrum of ferricytochrome c-556 contains the elements of two ferric iron species, one a low spin and the other a high spin form.The function of cytochrome c-556 is obscure. The purified cytochrome does not react with Pseudomonas cytochrome oxidase nor with the Pseudomonas cytochrome c-551 or copper protein.The properties of cytochrome c-556 indicate that it is probably not the same species as the cytochrome c-554 previously isolated from the same organism.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

Preliminary Studies of Fluorescent Pseudomonads Capable of Growth at 41 C in Swimming Pool Waters

During the summer of 1973 cultures of Pseudomonas aeruginosa and other fluorescent pseudomonads capable of growth at 41 C were obtained from swimming pool waters at a training center for the mentally retarded. Isolates were subjected to selected physiological tests, pyocine typing, and immunotyping. High counts of P. aeruginosa or other fluorescent pseudomonads consisted mainly of single predominant types. Both P. aeruginosa strains and unidentified fluorescent Pseudomonas strains predominated in pool waters at different times.     

Selection of Pseudomonas for industrial textile dyes decolourization

Pigment is the first contaminant to be recognised in bodies of water and wastewater. Besides the aesthetic problem, dyes obstruct light and reduce oxygen mass transfer. This paper describes the selection of Pseudomonas strains with the ability to remove colour from textile industrial dyes. Four Pseudomonas species were tested against 14 commercial industrial dyes. Pseudomonas cepacia exhibited no growth at all on plates containing dyes (1 g l^?1), whereas Pseudomonas aeruginosa, Pseudomonas oleovorans and Pseudomonas putida exhibited considerable growth. Decolourization in a liquid culture revealed that P. oleovorans is more viable for decolourizing textile dyes, as it achieved over 80% colour removal for two of the 14 dyes studied; it also proved to be more tolerant to high dye concentrations.     

Bioefficacy of some Rhizobactrial isolates against sorghum root Rot pathogen Bipolaris sorokiniana

This study carried out to identify certain microbial allelochemicals with antifungal activity of some rhziobacterial isolates against Bipolaris sorokiniana fungi. The fungicidal activity of isolated microbe metabolites was compared based on inhibition % of fungal growth. Results showed that ethyl acetate crude extracts with two concentrations (500 and 1000 ppm) of Pseudomonas geniculata (SC) and Bacillus cereus (S4) were the most efficient isolates recorded inhibition % 33.62 and 52.59% followed by S4 (Bacillus cereus (ATCC 14579) which achieved inhibition % 33.62 and 46.55% at the same concentrations, respectively. After 4 days.The constituents analyzed by LC-MS/MS and FTIR of the ethyl acetate extracts of the Pseudomonas geniculata ATCC19374 were afforded aminobutyric acid, 1,4-benzoquinone, coumaric acid, sinapic acid, tryptophan amino acid, Succinic acid and ferulic acid. While, the secondary metabolites of (Bacillus cereus ATCC 14579 extract were aminobutyric acid, 1,4-benzoquinone, coumaric acids, sinapic acid, ferulic acid and benzoic acid. Results indicated that the isolates of Pseudomonas geniculata ATCC19374 and Bacillus cereus ATCC 14579 could be use as a good element in plant root rot pathogen Bipolaris sorokiniana management.     

Mineralization of Paraoxon and Its Use as a Sole C and P Source by a Rationally Designed Catabolic Pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into β-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.     

Improved PCR for identification of Pseudomonas aeruginosa

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41?×?10³?copies/μl of cloned amplified target DNA using purified DNA, or 2.7?×?10² colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.     

Degradation of tetradecyltrimethylammonium by Pseudomonas putida A ATCC 12633 restricted by accumulation of trimethylamine is alleviated by addition of Al 3+ ions

Aims: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis’ acid in the medium influences its biodegradability. Methods and Results: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l^?1 of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0·1 mmol l^?1 AlCl₃ added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al³⁺ : TMA complex. Conclusions: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl₃. Significance and Impact of the Study: The use of Lewis’ acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.