Risk factors and antimicrobial resistance profiles of Pseudomonas putida infection in Central China, 2010-2017.

The aim of this study was to analyze the risk factors, clinical features, and antimicrobial resistance of Pseudomonas putida (P putida) isolated from Tongji Hospital in Wuhan, China.The data of 44 patients with P putida infections were retrospectively reviewed in this study. All cases of P putida strains were detected by the clinical laboratory of Tongji Hospital in the period of January 2010 to December 2017. Antimicrobial susceptibility testing was conducted using Kirby-Bauer method.Forty-four effective strains of P putida were isolated, including 32 inpatients and 12 outpatients. The 32 inpatients cases were obtained from various departments, which were urosurgery wards (n = 5, 15.6%), pediatrics wards (n = 4, 12.5%), hepatic surgery wards (n = 4, 12.5%), among others. The isolates had been discovered from urine specimens (28.2%), blood specimens (21.9%), sputum specimens (12.5%), and so on. Twenty-five patients had histories of catheterization before the isolation of P putida. Twenty-four patients were in immunocompromised states, 5 patients had undergone surgery, catheterization and were taking immunosuppressive therapy simultaneously. Polymicrobial infections were found in some P putida cases, especially Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Escherichia coli. All the patients had treated by antimicrobial before culture. Multi-drug-resistant strains were detected in 75% of P putida isolates. The P putida strains were resistant to trimethoprim/sulfamethoxazole (97.7%), aztreonam (88.6%), minocyline (74.3%), ticarcillin/clavulanic acid (72.7%), and sensitive to amikacin (86.4%), imipenem (62.8%), gentamicin (56.8%).Catheterization or other invasive procedures, immunocompromised states, and underlying diseases increased the risks of P putida infections. Moreover, the P putida strains were highly resistant to trimethoprim/sulfamethoxazole, aztreonam, minocyline, ticarcillin/clavulanic acid.

Construction and optimization of a Pseudomonas putida whole-cell bioreporter for detection of bioavailable copper.

A Pseudomonas putida whole-cell bioreporter for detecting bioavailable copper was constructed by inserting a CueR-regulated sensor element upstream of a promoterless green fluorescent protein (GFP) reporter gene. The constructed bioreporter cells expressed GFP only in response to Cu and Ag when cultivated in different metal salt solutions. M9 supplemented medium provided higher sensitivity compared with LB medium. The optimal test condition was cell suspension with an OD600 of 0.4-0.5 incubated at 30 °C. The detection range of Cu is 1-70 mg/l under optimal test condition in M9 supplemented medium.

Selected antimicrobial essential oils eradicate Pseudomonas spp. and Staphylococcus aureus biofilms.

Biofilms are difficult to eliminate with standard antimicrobial treatments due to their high antibiotic resistance relative to free-living cells. Here, we show that selected antimicrobial essential oils can eradicate bacteria within biofilms with higher efficiency than certain important antibiotics, making them interesting candidates for the treatment of biofilms.

Flavonoid glucuronides and a chromone from the aquatic macrophyte Stratiotes aloides.

The first phytochemical analysis of the aquatic macrophyte Stratiotes aloides afforded two new flavonoid glucuronides, luteolin 7-O-beta-D-glucopyranosiduronic acid-(1-->2)-beta-D-glucopyranoside (1) and chrysoeriol 7-O-beta-D-glucopyranosiduronic acid-(1-->2)-beta-D-glucopyranoside (2), as well as the new 2-(2-hydroxypentyl)-5-carboxy-7-methoxychromone (5) and chrysoeriol 7-O-beta-(6-O-malonyl)glucopyranoside (3), which has been assigned via NMR data for the first time. Additionally, free amino acids such as tryptophan, arginine, leucine, isoleucine, phenylalanine, and tyrosine along with choline, cis-aconitic acid, the phenolic glycoside alpha-arbutine, the chlorophyll derivative phaeophorbide a, and the flavonoid glycoside luteolin 7-O-beta-(6-O-malonyl)glucopyranoside (4) were isolated. Despite the low quantities obtained in some cases (between 50-300 microg), the structures of all compounds were unambiguously elucidated by extensive NMR and MS experiments. With a delay of 2 days compound 1 (10 and 50 microM test concentration) strongly inhibited the growth of human SH-SY5Y neuroblastoma cells in a dose-dependent manner, whereas only a moderate growth inhibition of human Patu 8902 carcinoma cells could be observed. Compounds 1 and 2 showed no activities against the bacteria Escherichia coli BW25113, Pseudomonas pudida KT2440, and Enterobacter cloacae subsp. dissolvens.

Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components.

OBJECTIVES: Thirty isolates of Pseudomonas aeruginosa, 15 isolates of Pseudomonas putida and 11 isolates of Pseudomonas fluorescens were tested for susceptibility to tea tree oil (TTO), the essential oil of Melaleuca alternifolia, and the components terpinen-4-ol, alpha-terpineol, cineole, gamma-terpinene and rho-cymene. METHODS: MICs were determined by broth microdilution in Mueller-Hinton medium supplemented with 0.002% (v/v) Tween 80. RESULTS: The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species. CONCLUSIONS: Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.

Biodegradation of p-nitrophenol by P. putida.

A strain of Pseudomonas putida was found capable of metabolizing p-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. To explore the applicability of this strain for bioremediation for controlling environmental PNP pollution, its degradation potential at 300 and 500 ppm was examined in a medium devoid of carbon and nitrogen source (minimal medium). At A600, 0.5 OD inoculum, the strain metabolized 300 and 500 ppm within 36 and 72 h, respectively. The degradation was accompanied by release of stoichiometric amount of nitrite. Effect of glucose and nitrogen on PNP degradation under similar conditions revealed that (i) glucose (0.4 g/l) at 20 and 50 ppm PNP did not accelerate the rate of PNP degradation, while glucose (0.4 g/l) at 300 ppm PNP inhibited its degradation, (ii) nitrogen supplement viz. sodium nitrate and ammonium sulphate (0.04 and 0.4 g/l) in minimal medium with PNP (300 ppm) showed no effect on PNP degradation, while glutamate alone (0.04 and 0.4 g/l) showed mere rise in biomass (from 0.5 to 1.6 OD units), and (iii) acidic pH (4.0-6.5) did not support PNP degradation, while alkaline pH (7.5-9.5) significantly enhanced the rate of PNP degradation. The complete degradation of PNP at high concentration (300 ppm) was confirmed by HPTLC analysis. In order to probe root cause of higher PNP degradation, preliminary studies on genetic analysis of P. putida were undertaken, which revealed the prevalence of a degradative plasmid of approximately 15 kb, while cured derivatives of P. putida (PNP-) did not show ability to degrade PNP. Further conjugal transfer of PNP+ phenotype from P. putida to standard strain of E. coli Nova blue (PNP-) confirmed the degradative type of plasmid.

Incorporating zosteric acid into silicone coatings to achieve its slow release while reducing fresh water bacterial attachment.

Biofouling has posed serious problems in maritime industry including increased fuel consumptions, economic loss from ship-hull maintenances, contamination of drinking water, and serious corrosion for mechanical instruments. Minimizing the attachment of bacteria and formation of biofilm could be advantageous in reducing the early stages of biofouling. Zosteric acid, a natural product present in eelgrass, was found to have ability for preventing the attachment of some bacteria and barnacles. In this study, the antifouling ability of zosteric acid during the early stages of fouling was evaluated using attachment studies of fresh water bacteria. Simultaneously, various methods were sought for incorporating zosteric acid into silicone to prolong the release of the compound. The main results from this study were that zosteric acid exhibited anti-bacterial attachment regardless of whether it dispersed in water or incorporated into a coating. In addition, the release rate of zosteric acid from the incorporated coatings, particularly those where zosteric acid was uniformly dispersed with aggregates size of 4 microm or less, was orders of magnitude slower than those of previous reports. The release results indicate that the service life of our coatings could be far extended even with a small amount of zosteric acid incorporated.

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed.

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.

Pseudomonas putida mutants in the exbBexbDtonB gene cluster are hypersensitive to environmental and chemical stressors.

The genes in the exbBexbDtonB cluster of Pseudomonas putida DOT-T1E are co-transcribed. We have generated non-polar mutants in each of the genes by inserting an aphA3 cassette encoding kanamycin resistance. All three mutants show similar phenotypes: the mutants are unable to grow on minimal medium under iron deficiency conditions. Furthermore, regardless of iron conditions, all mutants are hypersensitive to antibiotics, p-hydroxybenzoate and toluene, chemicals that are extruded from the cell by efflux pumps. These findings are discussed in terms of the involvement of the TonB system in the energization of outer membrane functions necessary for the import or export of different compounds in P. putida.

Recombinant carbazole-degrading strains for enhanced petroleum processing.

Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds. Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning. In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel. The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp. LD2, in a solvent-resistant heterologous host, P. putida Idaho [1]. This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp. LD2. Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations. Recombinant P. putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase. Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value.

Activation of soil respiration and shift of the microbial population balance in soil as a response to Lavandula stoechas essential oil.

Lavandula stoechas, a native plant of Greece, is rich in essential oil and fenchone is its major constituent. We examined the effect of the essential oil and its main constituents on soil metabolism and microbial growth. Addition of the essential oil or fenchone to soil samples induced a remarkable increase in soil respiration. This was accompanied by an increase in the soil bacterial population of three orders of magnitude. This sizable population was not qualitatively similar to that of the control soil samples. One bacterial strain dominated soil samples treated with L. stoechas essential oil or fenchone. By use of the disk diffusion assay, we evaluated the capacity of three bacterial strains that we isolated from the soil samples, as well as Escherichia coli and Bacillus subtilis (reference strains), to grow in the presence of the essential oil and three of its main constituents (fenchone, cineol, alpha-pinene). The substances tested did not inhibit the growth of the strain found to dominate the bacterial populations of treated soil samples; they severely inhibited B. subtilis. The other two isolated strains could also grow in liquid cultures in the presence of different quantities of essential oil or fenchone. Addition of fenchone at the end of the exponential phase increased the cell numbers of the strain that dominated the bacterial populations of treated soil samples, indicating use of the substrate added. On the basis of these results, we propose a scheme of successional stages during the decomposition process of the rich-in-essential-oil litter of aromatic plants that abound in the Mediterranean environment.

Characterization and expression of the pseudomonas putida bacterioferritin alpha subunit gene.

The root-colonizing pseudomonad Pseudomonas putida (Pp) appears to produce two subunits, alpha and beta, of the iron-binding protein, bacterioferritin. A gene encoding the alpha-bacterioferritin subunit was located adjacent to the major catalase in Pp. The deduced protein sequence of the Pp bfralpha gene had a very high identity with other alpha-subunits, possessing conserved amino acids responsible for ferroxidase activity. The gene also lacked a deduced methionine at residue 52, associated with heme binding in beta-subunits. An antibody generated toward the Escherichia coli (E. coli) multifunctional single subunit bacterioferritin recognized two proteins in the Pp extract, a 22 kDa protein likely to be a beta-subunit and, to a lesser extent, a 23 kDa band. The 23 kDa band was absent in a Pp mutant in which the bfralpha gene was disrupted. Loss of alpha-bacterioferritin stimulated production of fluorescent siderophore. Growth on media and on root surfaces was not impaired by deletion of the alpha-bacterioferritin. Transcription of bfralpha was independent of the catalase gene and was dependent on iron. The transcript levels from bfralpha decreased in iron deficiency experienced during stationary-phase or upon treatment during growth with an iron chelator.

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates.

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.

Short-run tests for determining harmful effects of PCB-containing engine oils on cells.

Our main objective was to set up reproducible methods for a rapid determination of harmful effects of PCB-containing engine oils on cells. We used a plate method and Scenedesmus quadricauda, Saccharomyces cerevisiae, Rhodotorula glutinis and Pseudomonas putida as test organisms.