Isolation and characterization of nutrient dependent pyocyanin from Pseudomonas aeruginosa and its dye and agrochemical properties.

Pyocyanin is a blue green phenazine pigment produced in large quantities by active cultures of Pseudomonas aeruginosa, with advantageous applications in medicine, agriculture and for the environment. Hence, in the present study, a potent bacterium was isolated from agricultural soil and was identified morphologically and by 16S rRNA sequencing as P. aeruginosa (isolate KU_BIO2). When the influence of nutrient supplements in both King's A and Nutrient media as amended was investigated, an enhanced pyocyanin production of 2.56 µg ml-1 was achieved in King's A medium amended with soya bean followed by 1.702 µg ml-1 of pyocyanin from the nutrient medium amended with sweet potato. Purified pyocyanin was characterized by UV-Vis Spectrophotometer and Fourier-Transform Infrared spectroscopy (FTIR). Furthermore, Liquid Chromatography Mass Spectrum (LCMS) and Nuclear Magnetic Resonance (NMR) confirmed its mass value at 211 and as N-CH3 protons resonating at 3.363 ppm as a singlet respectively. The isolated pyocyanin displayed remarkable dye property by inducing color change in cotton cloth from white to pink. Lastly, the antifungal activity of test pyocyanin showed inhibition of growth of rice blast fungus, Magnaporthe grisea and bacterial blight of rice, Xanthomonas oryzae at concentrations of 150 and 200 ppm, respectively. Thus, this investigation provides evidence for diverse actions of pyocyanin which are nutrient dependent and are capable of acting on a large scale, by utilizing microbes existing in agriculture wastes, and thus could be used as an alternative source in the making of natural textile dyes with strong durability and a broad spectrum of ecofriendly agrochemicals.

Surface-Enhanced Raman Scattering Spectroscopy for Label-Free Analysis of P. aeruginosa Quorum Sensing.

Bacterial quorum sensing systems regulate the production of an ample variety of bioactive extracellular compounds that are involved in interspecies microbial interactions and in the interplay between the microbes and their hosts. The development of new approaches for enabling chemical detection of such cellular activities is important in order to gain new insight into their function and biological significance. In recent years, surface-enhanced Raman scattering (SERS) spectroscopy has emerged as an ultrasensitive analytical tool employing rationally designed plasmonic nanostructured substrates. This review highlights recent advances of SERS spectroscopy for label-free detection and imaging of quorum sensing-regulated processes in the human opportunistic pathogen Pseudomonas aeruginosa. We also briefly describe the challenges and limitations of the technique and conclude with a summary of future prospects for the field.

Amplification of electrochemical signal by a whole-cell redox reactivation module for ultrasensitive detection of pyocyanin.

A bioelectrochemical sensing system based on a novel whole-cell redox reactivation/cycling module for ultrasensitive detection of pyocyanin (the biomarker of Pseudomonas aeruginosa infections) was developed. The electroactive bacteria mediated redox reactivation module was constructed using Shewanella oneidensis MR-1 cells as the bioelectro-catalyst and lactate as the electron donor. It could regenerate reductive pyocyanin from its oxidative state, which enabled pyocyanin molecule repeatedly registered by the electrode. Uniquely, with this redox reactivation module, the electrochemical response of pyocyanin was amplified about 405 times (1.3 µA/nM vs. 3.2nA/nM). Thus, an ultrasensitive bioelectrochemical sensing system for pyocyanin quantification was developed by integrating the pyocyanin reactivation module with conventional electrochemical detection system. Remarkably, with this developed biosensing system, an extremely low LOD of 47±1pM was reached. Additionally, this biosensing system showed excellent resistance to interferences from human fluids or bacterial contamination. This work provided a simple, ultrasensitive and robust tool for pyocyanin detection, and more importantly, demonstrated a new dimension for electrochemical signal amplification in biosensing.

Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry.

Pyocyanin is a virulence factor uniquely produced by the pathogen Pseudomonas aeruginosa. The fast and selective detection of pyocyanin in clinical samples can reveal important information about the presence of this microorganism in patients. Electrochemical sensing of the redox-active pyocyanin is a route to directly quantify pyocyanin in real time and in situ in hospitals and clinics. The selective quantification of pyocyanin is, however, limited by other redox-active compounds existing in human fluids and by other metabolites produced by pathogenic bacteria. Here we present a direct selective method to detect pyocyanin in a complex electroactive environment using commercially available electrodes. It is shown that cyclic voltammetry measurements between -1.0 V to 1.0 V reveal a potential detection window of pyocyanin of 0.58-0.82 V that is unaffected by other redox-active interferents. The linear quantification of pyocyanin has an R² value of 0.991 across the clinically relevant concentration range of 2-100 µM. The proposed method was tested on human saliva showing a standard deviation of 2.5% ± 1% (n = 5) from the known added pyocyanin concentration to the samples. This inexpensive procedure is suggested for clinical use in monitoring the presence and state of P. aeruginosa infection in patients.

Pyocyanin: production, applications, challenges and new insights.

Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium and is one of the most commercially and biotechnologically valuable microorganisms. Strains of P. aeruginosa secrete a variety of redox-active phenazine compounds, the most well studied being pyocyanin. Pyocyanin is responsible for the blue-green colour characteristic of Pseudomonas spp. It is considered both as a virulence factor and a quorum sensing signalling molecule for P. aeruginosa. Pyocyanin is an electrochemically active metabolite, involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognised as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. This review summarises recent advances of pyocyanin production from P. aeruginosa with special attention to antagonistic property and bio-control activity. The review also covers the challenges and new insights into pyocyanin from P. aeruginosa.

Exploring antagonistic metabolites of established biocontrol agent of marine origin.

Biocontrol ability of Pseudomonas aeruginosa ID 4365, a biocontrol agent of groundnut phytopathogens from marine origin, was previously attributed to the production of pyoverdin type of siderophores. However, pyoverdin-rich supernatants of this organism showed better antifungal activity compared to equivalent amount of purified pyoverdin indicating presence of undetected metabolite(s) in pyoverdin rich supernatants. On the basis of observation that antagonistic activity was iron-dependent and iron-independent, an attempt was made to detect the presence of additional metabolites. In addition to pyoverdin, strain produced additional siderophores, viz. pyochelin and salicylic acid. Two broad spectrum antifungal compounds, viz. pyocyanin and phenazine-1-carboxylic acid, were detected, characterized, and activity against phytopathogens was demonstrated. Iron- and phosphate-dependent co-production of siderophores and phenazines was confirmed. Strain showed additional features like production of hydrogen cyanide, indol-3-acetic acid, and phosphate solubilization.

Pyocyanin isolated from a marine microbial population: synergistic production between two distinct bacterial species and mode of action.

Marine microbial populations collected from the Hawaiian Islands were screened for antimicrobial activity. A blue metabolite was identified from mixed cell cultures, but production was not evident in pure cultures. Experiments designed to probe the synergistic role of the microorganisms are presented. Full characterization of the blue natural product, pyocyanin, is provided including corrections made to 1H and 13C-NMR assignments of the molecule misreported in the chemical literature and yeast transcriptome analysis. The transcriptional effects were consistent with the compound's purported role as an inducer of oxidative stress and damage and illustrates the overall potential of the method to reveal the primary biological/cellular effects of a natural product. The experiments outlined here might serve as a general paradigm for identification of natural products arising from microbial communities and investigation of their respective interactions.

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa.

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth.

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.

A study of purified pyocyanine produced by Pseudomonas aeruginosa: properties and grouping.

A study of more than one thousand strains of Pseudomonas aeruginosa was performed. These were collected from different sources, i.e. humans, animals, environment and food. Cumulative results revealed the existence of three different groups of pyocyanine. The biological importance of grouping this pigment is due to its activity against other bacteria in comparison with other antibiotics such as cyanomycin produced by Streptomyces cyanoflavus. No such grouping or any other classification was found in the literature. Stability test and MIC (minimum inhibitory concentration) measurements revealed the priority of group (I); also isolates of animal origin were found preferable. This may be due to the resistance of strains isolated from animals to antibiotics especially to carbenicillin and gentamycine. Observations indicate differences in optical properties of the blue pigment, i.e. absorption centres in the UV region. Some differences in their physical properties were also noted.

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.

Medium for the simultaneous detection of pyocyanin and fluorescein pigments of Pseudomonas aeruginosa.

To help simplify the identification of Pseudomonas aeruginosa by clinical microbiology laboratories, the authors developed a new medium, pyocyanin-fluorescein agar (PFA), which enhances the production of both Pseudomonas pigments simultaneously. Production of pigments on PFA was equivalent to production on a pyocyanin agar (P agar) and a fluorescein agar (F agar) used in combination and was superior to either P agar or F agar used alone. The medium is simple to prepare and it detected pigment in 94% of P. aeruginosa isolates tested.

Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro.

Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

Purification and structural analysis of pyocyanin and 1-hydroxyphenazine.

Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance. Pyocyanin and its base hydrolysis product 1-hydroxyphenazine were purified to homogeneity by reverse-phase high-pressure liquid chromatography. Their mass spectrometric behaviour was examined with a view to evaluating the use of high-resolution chromatography/mass spectrometry in studying phenazine-mediated effects in man. The molecular mass of naturally derived pyocyanin was determined as 210 Da by thermospray liquid chromatography/mass spectrometry and confirmed by desorption electron-impact mass spectrometry. Mass spectrometric data could not be obtained by fast-atom bombardment or desorption chemical ionisation, techniques commonly used to determine molecular mass of polar or thermally labile species. The thermal lability of underivatised pyocyanin precluded analysis by gas chromatography/mass spectrometry. In contrast to pyocyanin, mass spectrometric data were readily obtained for 1-hydroxyphenazine, using direct probe analysis as well as with gas and liquid chromatography inlet systems.

Pyocyanin preparation from Pseudomonas aeruginosa isolated from heterogeneous clinical materials.

Pure pyocyanin was prepared from 120 strains of Pseudomonas aeruginosa isolated from different clinical specimens. The modified method used increased the purification 425-fold. The spectra of pyocyanin of all strains were found to be identical with a single major peak at 363 nm. The only difference between these spectra appeared to be the level value absorbance. Strains recovered from different clinical specimens differ in their ability for pigment production. The strains isolated from urine produced higher amounts of pyocyanin than other isolates.