Electrochemical detection of Pseudomonas in wound exudate samples from patients with chronic wounds.

In clinical practice, point-of-care diagnostic testing has progressed rapidly in the last decade. For the field of wound care, there is a compelling need to develop rapid alternatives for bacterial identification in the clinical setting, where it generally takes over 24 hours to receive a positive identification. Even new molecular and biochemical identification methods require an initial incubation period of several hours to obtain a sufficient number of cells prior to performing the analysis. Here we report the use of an inexpensive, disposable electrochemical sensor to detect pyocyanin, a unique, redox-active quorum sensing molecule released by Pseudomonas aeruginosa, in wound fluid from patients with chronic wounds enrolled in the WE-HEAL Study. By measuring the metabolite excreted by the cells, this electrochemical detection strategy eliminates sample preparation, takes less than a minute to complete, and requires only 7.5 µL of sample to complete the analysis. The electrochemical results were compared against 16S rRNA profiling using 454 pyrosequencing. Blind identification yielded 9 correct matches, 2 false negatives, and 3 false positives giving a sensitivity of 71% and specificity of 57% for detection of Pseudomonas. Ongoing enhancement and development of this approach with a view to develop a rapid point-of-care diagnostic tool is planned.

Electrochemically monitoring the antibiotic susceptibility of Pseudomonas aeruginosa biofilms.

The condition of cells in Pseudomonas aeruginosa biofilms was monitored via the electrochemical detection of the electro-active virulence factor pyocyanin in a fabricated microfluidic growth chamber coupled with a disposable three electrode cell. Cells were exposed to 4, 16, and 100 mg L(-1) colistin sulfate after overnight growth. At the end of testing, the measured maximum peak current (and therefore pyocyanin concentration) was reduced by approximately 68% and 82% in P. aeruginosa exposed to 16 and 100 mg L(-1) colistin sulfate, respectively. Samples were removed from the microfluidic chamber, analyzed for viability using staining, and streaked onto culture plates to confirm that the P. aeruginosa cells were affected by the antibiotics. The correlation between electrical signal drop and the viability of P. aeruginosa cells after antibiotic exposure highlights the usefulness of this approach for future low cost antibiotic screening applications.

Up-regulating pyocyanin production by amino acid addition for early electrochemical identification of Pseudomonas aeruginosa.

This work focuses on developing a faster method for electrochemically detecting a Pseudomonas aeruginosa infection through the addition of amino acids to cell culture samples. We performed square-wave voltammetry measurements of pyocyanin produced by P. aeruginosa using commercially available carbon-based electrodes connected to a Ag/AgCl reference. The electrochemical response resulting from the production of pyocyanin by bacteria was measured in the presence of various amino acids while varying three different culturing parameters: liquid media type (trypticase soy broth vs. M63 minimal media); concentration of amino acids in the solution; and initial concentration of the P. aeruginosa in the solution. Our results demonstrate a faster and stronger electrochemical response in media containing tyrosine and valine at elevated concentrations, lending promise to using amino acids as up-regulatory molecules for faster bacterial detection.

Electrochemical Probes of Microbial Community Behavior.

Advances in next-generation sequencing technology along with decreasing costs now allow the microbial population, or microbiome, of a location to be determined relatively quickly. This research reveals that microbial communities are more diverse and complex than ever imagined. New and specialized instrumentation is required to investigate, with high spatial and temporal resolution, the dynamic biochemical environment that is created by microbes, which allows them to exist in every corner of the Earth. This review describes how electrochemical probes and techniques are being used and optimized to learn about microbial communities. Described approaches include voltammetry, electrochemical impedance spectroscopy, scanning electrochemical microscopy, separation techniques coupled with electrochemical detection, and arrays of complementary metal-oxide-semiconductor circuits. Microbial communities also interact with and influence their surroundings; therefore, the review also includes a discussion of how electrochemical probes optimized for microbial analysis are utilized in healthcare diagnostics and environmental monitoring applications.

Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates.

In clinical practice, delays in obtaining culture results impact patient care and the ability to tailor antibiotic therapy. Despite the advancement of rapid molecular diagnostics, the use of plate cultures inoculated from swab samples continues to be the standard practice in clinical care. Because the inoculation culture process can take between 24 and 48h before a positive identification test can be run, there is an unmet need to develop rapid throughput methods for bacterial identification. Previous work has shown that pyocyanin can be used as a rapid, redox-active biomarker for identifying Pseudomonas aeruginosa in clinical infections. However, further validation is needed to confirm pyocyanin production occurs in all clinical strains of P. aeruginosa. Here, we validate this electrochemical detection strategy using clinical isolates obtained from patients with hospital-acquired infections or with cystic fibrosis. Square-wave voltammetric scans of 94 different clinical P. aeruginosa isolates were taken to measure the concentration of pyocyanin. The results showed that all isolates produced measureable concentrations of pyocyanin with production rates correlated with patient symptoms and comorbidity. Further bioinformatics analysis confirmed that 1649 genetically sequenced strains (99.9%) of P. aeruginosa possess the two genes (PhzM and PhzS) necessary to produce pyocyanin, supporting the specificity of this biomarker. Confirming the production of pyocyanin by all clinically-relevant strains of P. aeruginosa is a significant step towards validating this strategy for rapid, point-of-care diagnostics.

Electrochemical detection of Pseudomonas aeruginosa in human fluid samples via pyocyanin.

The ability to quickly detect the presence of pathogenic bacteria in patient samples is of the outmost importance to expedient patient care. Here we report the direct, selective, and sensitive detection of the opportunistic pathogen Pseudomonas aeruginosa, spiked in human whole blood with sodium heparin, urine, sputum, and bronchial lavage samples using unmodified, disposable carbon electrode sensors that detect the presence of pyocyanin, a virulence factor that is unique to this species. Square wave voltammetry scans of biological fluids from healthy individuals spiked with P. aeruginosa showed a clear pyocyanin response within one day of culturing at 37°C. Scans of supernatants taken from cultures of P. aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, and Bacillus cereus taken over a span of three days in the potential range from -0.5 to 0 V vs. an Ag/AgCl reference showed no electrochemically detectable molecules with the exception of P. aeruginosa. The results indicate the potential to sensitively and selectively determine the presence of P. aeruginosa in human samples via the electrochemical detection of pyocyanin in less than 5 min, without any sample preparation or separation steps.