铜绿假单胞菌Arr基因突变对生物膜和绿脓菌素合成的影响

为了研究铜绿假单胞菌Arr基因对生物膜和绿脓菌素合成的影响,采用抗庆大霉素基因序列(Gentamycin resistance cassette,aacC1)插入失活的策略构建了铜绿假单胞菌Arr基因突变株PA-AG,通过96孔板静止培养、结晶紫染色的方法检测其生物膜的形成量,利用抽提的方法检测绿脓菌素的合成量。结果在KMB或LB培养基中,突变株PA-AG形成生物膜的量均有所减少,野生株约是突变株的2倍,然而突变株合成绿脓菌素的能力却明显加强,约为野生株的2.5倍。由此推测,铜绿假单胞菌Arr基因在一定程度上促进了生物膜的形成,抑制了绿脓菌素的合成。     

Pseudomonas aeruginosa (GRC1) as a strong antagonist of Macrophomina phaseolina and Fusarium oxysporum

A plant growth promotory bacterial strain, isolated from the potato rhizosphere, was characterized as Pseudomonas aeruginosa (GRC1). The isolate produced an hydroxamate type of siderophore after 48 h of incubation on tryptic soy medium under iron deficient conditions. The in vitro antifungal activity of P. aeruginosa was tested against two soil-borne plant pathogens, Macrophomina phaseolina and Fusarium oxysporum. The antagonistic behaviour of the isolate was tested by dual culture technique. The growth inhibition of M. phaseolina and F. oxysporum was 74.1% and 70.5%, respectively, after 5 days of incubation. The production of hydrocyanic acid and indole acetic acid was also recorded under normal growth conditions.     

7-fluoroindole as an antivirulence compound against Pseudomonas aeruginosa

The emergence of antibiotic resistance has necessitated new therapeutic approaches for combating persistent bacterial infection. An alternative approach is regulation of bacterial virulence instead of growth suppression, which can readily lead to drug resistance. The virulence of the opportunistic human pathogen Pseudomonas aeruginosa depends on a large number of extracellular factors and biofilm formation. Thirty-one natural and synthetic indole derivatives were screened. 7-fluoroindole (7FI) was identified as a compound that inhibits biofilm formation and blood hemolysis without inhibiting the growth of planktonic P.?aeruginosa cells. Moreover, 7FI markedly reduced the production of quorum-sensing (QS)-regulated virulence factors 2-heptyl-3-hydroxy-4(1H)-quinolone, pyocyanin, rhamnolipid, two siderophores, pyoverdine and pyochelin. 7FI clearly suppressed swarming motility, protease activity and the production of a polymeric matrix in P.?aeruginosa. However, unlike natural indole compounds, synthetic 7FI did not increase antibiotic resistance. Therefore, 7FI is a potential candidate for use in an antivirulence approach against persistent P.?aeruginosa infection.     

Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.     

N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa

Acinetobacter baumannii and Pseudomonas aeruginosa are pathogens capable of colonizing the same infection sites and employing N-acyl homoserine lactone (AHL) based quorum-sensing systems to co-ordinate biofilm formation. Hence, the effect of P. aeruginosa AHLs on biofilm formation by A. baumannii and vice versa were investigated using the biofilm impaired quorum sensing mutants, A. baumannii M2 (abaI::Km) and P. aeruginosa PAO-JP2. Complementing the mutants with heterologous, extracted and pure AHLs increased biofilm mass significantly. The surface area coverage and biovolume also increased significantly as observed by confocal scanning laser microscopy which corroborated scanning electron microscope analysis. Autoinducer synthase gene promoters of A. baumannii, P( abaI)-lacZ, and P. aeruginosa, P( lasI)-lacZ, were induced (p?相似文献   

Beneficial effects of fluorescent pseudomonads on seed germination, growth promotion, and suppression of charcoal rot in groundnut (Arachis hypogea L.)

Rhizobacteria are used as inoculants to enhance crop yield and for biological control of fungal pathogens. Fluorescent pseudomonads isolated from the rhizosphere of groundnut showed suppression of the phytopathogen Macrophomina phaseolina that causes charcoal rot of groundnut, an economically important agroproduct. Two strains of fluorescent pseudomonads, designated as PS1 and PS2, were selected as a result of in vitro antifungal activity. After 5 days of incubation at 28+/-1 degrees , both PS1 and PS2 caused clear inhibition zones in dual cultures, restricting the growth of M. phaseolina by 71% and 74%, respectively. Both the strains were capable of producing siderophores, indole acetic acid, and hydrocyanic acid, and causing phosphate solubilization under normal growth conditions. These strains, when used as inoculants in groundnut, enhanced germination up to 15% and 30% with subsequent increase in grain yield by 66% and 77%, respectively. Conversely, when the pathogen alone was testeds 57% decrease in yield was recorded. Thus the studies revealed the potential of the two pseudomonads not only as biocontrol agents against M. phaseolina, but also as a good growth promoter for groundnut.     

The Pseudomonas toxin pyocyanin inhibits the dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells

The dual oxidase-thiocyanate-lactoperoxidase (Duox/SCN(-)/LPO) system generates the microbicidal oxidant hypothiocyanite in the airway surface liquid by using LPO, thiocyanate, and Duox-derived hydrogen peroxide released from the apical surface of the airway epithelium. This system is effective against several microorganisms that infect airways of cystic fibrosis and other immunocompromised patients. We show herein that exposure of airway epithelial cells to Pseudomonas aeruginosa obtained from long-term cultures inhibits Duox1-dependent hydrogen peroxide release, suggesting that some microbial factor suppresses Duox activity. These inhibitory effects are not seen with the pyocyanin-deficient P. aeruginosa strain PA14 Phz1/2. We show that purified pyocyanin, a redox-active virulence factor produced by P. aeruginosa, inhibits human airway cell Duox activity by depleting intracellular stores of NADPH, as it generates intracellular superoxide. Long-term exposure of human airway (primary normal human bronchial and NCI-H292) cells to pyocyanin also blocks induction of Duox1 by Th2 cytokines (IL-4, IL-13), which was prevented by the antioxidants glutathione and N-acetylcysteine. Furthermore, we showed that low concentrations of pyocyanin blocked killing of wild-type P. aeruginosa by the Duox/SCN(-)/LPO system on primary normal human bronchial epithelial cells. Thus, pyocyanin can subvert Pseudomonas killing by the Duox-based system as it imposes oxidative stress on the host. We also show that lactoperoxidase can oxidize pyocyanin, thereby diminishing its cytotoxicity. These data establish a novel role for pyocyanin in the survival of P. aeruginosa in human airways through competitive redox-based reactions between the pathogen and host.     

Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa

Chronic lung infection by mucoid Pseudomonas aeruginosa is one of the major pathologic features in patients with cystic fibrosis. Mucoid P.?aeruginosa is notorious for its biofilm forming capability and resistance to immune attacks. In this study, the roles of extracellular polymeric substances from biofilms formed by mucoid P.?aeruginosa were investigated. Alginate is not an essential structure component for mucoid P.?aeruginosa biofilms. Genetic studies revealed that Pel and Psl polysaccharides serve as essential scaffold and mediate macrocolony formation in mucoid P.?aeruginosa biofilms. The Psl polysaccharide is more important than Pel polysaccharide in mucoid P.?aeruginosa biofilm structure maintenance and phagocytosis resistance. The polysaccharides were further found to protect mucoid P.?aeruginosa strain from host immune clearance in a mouse model of acute lung infection.     

铜绿假单胞菌XQ17提取物对革兰阳性菌的拮抗作用

从临床分离的1株铜绿假单胞菌能够产生拮抗物质,对其粗提物的初步研究表明,能有效杀灭受试的多数革兰阳性菌。通过比较其基本特征,与已知的铜绿假单胞菌产生的其他抗菌物质如Pyocyanin、Phenazine-1-carboxamide、Pseudomonic acid、pyocin等有明显区别,提示可能是一种具有潜在应用价值的新抗菌物质。     

Pleiotropic effects of the mioC mutation on the physiology of Pseudomonas aeruginosa PAO1

Flavodoxin (Fld) is a bacterial electron-transfer protein that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation. Mutation of the mioC gene also influenced the production of pigments. The mioC mutant and mioC over-expressed complementation cells, over-produced pyocyanin and pyoverdine, respectively. Various secreted chemicals were also changed in the mutant, which was confirmed by (1) H NMR analysis. Interestingly, physiological alterations of the mutant strain were restored by the cell-free supernatant of the wild type. The present study demonstrates that the mioC gene plays an important role in the physiology of P.?aeruginosa and might be considered as a suitable drug target candidate in pathogenic P.?aeruginosa.     

Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits

AIM: To study the antifungal activity and plant beneficial traits of a broad-spectrum antagonistic fluorescent pseudomonad strain, PUPa3. METHODS AND RESULTS: Strain PUPa3 was isolated from the rhizosphere soil of rice and identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences. This bacterium exhibits a broad-spectrum antifungal activity towards phytopathogenic fungi. The antifungal metabolite by PUPa3 was extracted, purified and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Production of indole-3-acetic acid (IAA), siderophores, phosphatase and protease in PUPa3 was determined. Strain PUPa3 did not produce hydrogen cyanide, cellulase and pectinase. CONCLUSION: The antifungal metabolite produced by PUPa3 has been identified as phenazine-1-carboxamide (PCN) on the basis of NMR and MS data. Strain PUPa3 showed a broad-spectrum antifungal activity towards a range of phytopathogenic fungi. This bacterium also showed several plant growth-promoting traits but did not show the traits attributed to deleterious rhizobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the production of PCN as well as IAA for the first time by a saprophytic P. aeruginosa strain PUPa3. Because of the production of siderophore, growth hormone, protease and phosphatase and its innate fungicidal potential, this strain can be used as biofertilizer and antagonist against a range of phytopathogenic fungi that infect rice, groundnut, tobacco, chili, mango, sugarcane, tea, cotton and banana.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.     

Ginseng aqueous extract attenuates the production of virulence factors, stimulates twitching and adhesion, and eradicates biofilms of Pseudomonas aeruginosa

This study was carried out to examine the antimicrobial activity of the aqueous extract of Panax quinquefolius from North American ginseng (NAGE) root against Pseudomonas aeruginosa . The minimum inhibitory concentrations of reference and clinical isolates of Pseudomonas aeruginosa were measured by a standard agar-dilution method. At subinhibitory NAGE concentrations, the secretion of virulence factors, motility on agar, and adhesion to 96-well microplates were studied on the nonmucoid Pseudomonas aeruginosa O1 strain. At suprainhibitory concentrations, the activity of NAGE against mature biofilm complexes formed in the Calgary Biofilm Device and the Stovall flow cell were assessed. NAGE possessed an antibacterial activity against all the Pseudomonas aeruginosa strains at 1.25%-2.5% w/v. NAGE also significantly attenuated pyocyanin, pyoverdine, and lipase concentrations, stimulated twitching, and attenuated swarming and swimming motility. At 1.25% w/v, NAGE augmented adhesion, and at 5% w/v detached 1-day-old biofilms in microplates. The extract also eradicated 6-day-old mature biofilms (5% w/v), and fluorescence microscopy displayed a reduction of live cells and biofilm complexes compared with nontreated biofilms. These data suggest that the aqueous extract from North American ginseng possesses antimicrobial activities in vitro.     

The influence of mineral and carbon sources on biological control of charcoal rot fungus,Macrophomina phaseolina by fluorescent pseudomonads in tomato

AIMS: To determine the influence of various trace minerals and carbon source on the biocontrol performance of Pseudomonas aeruginosa strain IE-6S+ and P. fluorescens strain CHA0 against Macrophomina phaseolina. METHODS AND RESULTS: In dual culture plate assay, P. aeruginosa IE-6S+ and P. fluorescens CHA0 inhibited radial growth of M. phaseolina producing zones of inhibition. Czapek's dox agar medium amended with both zinc and glucose remarkably improved antifungal activities of the bacterial inoculants. Under glasshouse conditions, soil amendment with zinc and/or glucose alone did not reduce M. phaseolina infection in tomato roots but did reduce significantly when used in combination with IE-6S+ or CHA0. Soil amendments with zinc and/or glucose increased fresh shoot weights but zinc amendment greatly reduced bacterial populations in the rhizosphere. CONCLUSIONS: Mineral and carbon amendments enhance the biocontrol performance of fluorescent pseudomonads against M. phaseolina. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of mineral and carbon amendments that favour biocontrol of certain bacterial strains may provide clues to soil factors or components of nutrient solutions in hydroponic culture that will improve the level and reliability of control.     

椒样薄荷、薄荷和苏格兰留兰香精油与抗生素的协同抑菌功能

以开花期的椒样薄荷（Mentha×piperita）、薄荷（M.haplocalyx）和苏格兰留兰香（M.×gentilis）叶片部位提取的精油为研究对象,通过GC-MS分析,并采用纸片扩散法研究了3种精油单独使用及与抗生素联合使用时对金黄色葡萄球菌、蜡状芽孢杆菌、大肠杆菌、绿脓杆菌和肺炎克雷伯氏菌的抑制情况。结果表明,（1）椒样薄荷与薄荷精油中含量最高的成分为薄荷醇、薄荷酮和异薄荷酮,苏格兰留兰香精油的主要成分为香芹酮和柠檬烯。薄荷和苏格兰留兰香精油符合欧洲药典与ISO标准,椒样薄荷需要继续改良以提高其精油品质与抑菌功能。（2）精油单独使用时,Pseudomonas aeruginosa ATCC15442对椒样薄荷精油和薄荷精油敏感;P.aeruginosa ATCC27853对薄荷精油和苏格兰留兰香精油敏感。精油与抗生素联合使用时抑菌范围和强度均有所改变：绿脓杆菌的2个菌株对精油与抗生素的组合最为敏感,其中,椒样薄荷精油与头孢他啶的组合对P.aeruginosa ATCC15442显示出最强的增效作用,薄荷精油与头孢他啶混合之后对P.aeruginosa ATCC27853出现拮抗作用。Staphylococcus aureus ATCC25923对所有精油以及精油与抗生素混合物均有抗性。（3）椒样薄荷、薄荷和苏格兰留兰香精油的不同成分及其含量差异不仅对精油品质有影响,而且影响精油对测试菌种的抑制作用,可考虑将其作为薄荷属植物品质育种的参考指标。     

Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 microg per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.     

Pyocyanin production by Pseudomonas aeruginosa induces neutrophil apoptosis and impairs neutrophil-mediated host defenses in vivo

Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1beta) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.