Improved monitoring of P. aeruginosa on agar plates

Sensors

Pseudomonas aeruginosa is a ubiquitously distributed soil and water bacterium and is considered an opportunistic pathogen in hospitals. In cystic fibrosis patients, for example, infections with P. aeruginosa can be severe and often lead to chronic or even fatal pneumonia. Therefore, rapid detection and further identification are of major importance in hospital hygiene and infection control. This work shows the electrochemical properties of five P. aeruginosa key metabolites considering their potential use as specific signaling agents in an electrochemical sensor system. The pure solutes of pyocyanin (PYO), Pseudomonas quinolone signal (PQS), pyochelin (PCH), 2-heptyl-4-hydroxyquinoline (HHQ), and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) were analyzed by different electrochemical techniques (cyclic and square wave voltammetry) and measured using a Gamry Reference 600+ potentiostat. Screen-printed electrodes (DropSens DRP110; carbon working and counter, silver reference electrode) w...

Analytical and Bioanalytical Chemistry, 2019

The rapid detection of bacterial strains has become a major topic thoroughly discussed across the biomedical field. Paired with the existence of nosocomial pathogen agents that imply extreme medical and financial challenges throughout diagnosis and treatment, the development of rapid and easy-to-use sensing devices has gained an increased amount of attention. Moreover, antibiotic resistance considered by World Health Organization as one of the Bbiggest threats to global health, food security, and development today^enables this topic as high priority. Pseudomonas aeruginosa, one of the most ubiquitous bacterial strains, has various quorum-sensing systems that are a direct cause of their virulence. One of them is represented by pyocyanin, a blue pigment with electroactive properties that is synthesized from early stages of bacterial colonization. Thus, the sensitive detection of this biomarker could enable a personalized and efficient therapy. It was achieved with the development of an electrochemical sensor based on a thermosensitive polymer, modified with Au/Ag nanoalloy for the rapid and accurate detection of pyocyanin, a virulence biomarker of Pseudomonas aeruginosa. The sensor displayed a linear range from 0.12 to 25 μM, and a limit of detection of 0.04 μM (signal/noise = 3). It was successfully tested in real samples spiked with the target analyte without any pretreatment other than a dilution step. The detection of pyocyanin with high recovery in whole blood in a time frame of 5-10 min from the moment of collection was performed with this electrochemical sensor.

2017

Pseudomonas aeruginosa, a gram negative bacteria, which exerts broad antagonistic activity against other fungal pathogens through the production of a secondary metabolite were taken for P. aeruginosa isolation. From sixteen primary P. aeruginosa PP3) were selected, on the basis of pigmentation in cetrimide agar. The screening for antimicrobial activity of P aeruginosain cross streak method showed that Bacillus cereus (ATCC 14579) were sensitive, Escherichia coli (ATCC 8739) was intermediate, and K. pneumoniae (ATCC 43816) was resistant to the inhibitory action of the selected P. aeruginosa isolates. The antimicrobial pigment pyocyanin was extracted from culture broth following solvent extraction method. Purification of the pigment was done by column and thin layer chromatography. The Rf value was found around 0.81 for all the extracted pigment solution. Confirmation of the pigment as pyocyanin was done through FTIR, and UV functional groups (-OH, -C=N, -CH3 etc.) which belongs to th...

Sensors & Diagnostics

Despite bacterial biofilms representing a common form of infection, notably on medical devices post implantation, their detection and characterisation with existing methods is not sufficient to inform clinicians about biofilm...

2017

Objective(s): Pyocyanin is a blue-greenish redox-active pigment, produced by Pseudomonas aeruginosa, with a wide range of biological and biotechnological applications. Pyocyanin biosynthesis is regulated by the quorum-sensing (QS) system in which the expression of QS genes and QS-controlled virulence genes may be affected by serum as a complex medium. In the current study, effects of adult bovine serum (ABS) and fetal bovine serum (FBS) on the production of pyocyanin were examined in order to develop it. Materials and Methods: The presence of pyocyanin-producing specific genes and proteins in clinical and soil isolates of P. aeruginosa was confirmed using PCR and SDS-PAGE. Isolates were inoculated to media containing different concentrations of complement-active/-inactivated ABS or FBS and pyocyanin concentration was measured by spectrophotometry. Extracted pigment was characterized by using UV-Visible spectrophotometry. Titration of ABS antibodies against studied isolates was perfo...

MOJ Toxicology

Pyocyanin, the growth pigment produced by Pseudomonas aeruginosa, thought at one time to be a metabolic byproduct, was found to be important for the wellbeing of the organism and one of the important chemicals that allow it to survive in environments with competitive organisms. Different methods were used to extract and purify pyocyanin, some requiring major instruments. This study was performed to introduce an amended method for the optimal extraction and purification of pyocyanin. The results showed that the maximum yield was obtained from Cetrimide Agar plates seeded with the organism and held at room temperature for 96 hrs. The purified pyocyanin's rate of degradation was dependent on the concentration of the sodium hydroxide added, its optimal storage solvents were deionized water and/or 80% methanol, but not dry chloroform in which the pigment quickly degraded. The purified pyocyanin was found to be thermolabile as it was stable for many days at lower temperatures but was immediately inactivated by higher temperatures. The suggested method is easy to implement, requires only regular laboratory facilities and gives a good yield of the pure pigment. Recommendations are also provided for proper storage to maintain its activity for a considerable period of time.

PloS one, 2018

Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in ...

2015

P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of pyocyanin production was carried out in the different conditions of cultivation (temperature, carbon sources and salt concentrations) in a time-course manner. For the cultures grown at 30 °C and 1 % sucrose in the presence of 50 mM CaCl 2 , P. aeruginosa, 72 hours 4.53 mg/ml, PaJC is grown at 37 °C and 1 % sucrose in the presence of 250 mM CaCl 2 , 72 hours 3.96 mg/ml with pyocyanin producing reached the highest value. The nutrient source, especially the carbon type, had a dramatic effect on pyocyanin production. This pyocyanin producing with vgb may be an effective method.

Sensors and Actuators B: Chemical, 2004

Journal of Analysis and Testing, 2019

Impedance measurements using graphite electrodes were used to detect the increase in culture medium conductivity due to bacteria growth in real time along with simultaneous voltammetric monitoring of pyocyanin concentration. Electrochemical methods were compared to conventional continuous monitoring of bacterial cultures using turbidity measurement at an optical density of 600 nm (OD600). A practical osmotic system was further designed for concentrating bacterial cultures during growth to enable earlier detection using the electrochemical methods. Bacterial cultures, starting from an initial culture density of 1 × 10 8 cells/mL, were grown inside a sealed cellulose ester dialysis membrane, while polyethylene glycol in LB medium was used as the draw solution outside the membrane to gradually concentrate the growing cultures. 0.7-mm-diameter graphite for mechanical pencils was utilized as working and counter electrodes with a platinum wire reference electrode for electrochemical measurements with and without the osmotic system. In the absence of forward osmosis, impedance measurements detected culture growth ~ 1 h faster than conventional OD600. Integrating osmosis showed a twofold decrease in the time to detect pyocyanin production as an indicator for bacterial growth. For impedance monitoring, removal of water by osmosis was conflated with the impedance decrease due to cell growth; however, the results show a promising ability to detect bacteria growth via an observed shift in osmotic impedance profile when bacteria are present in a sample. By monitoring the deviation in the impedance profile, a threefold improvement in detection time was achieved when compared to conventional OD600 measurements.