Hunter J. Sismaet

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.

AMPEROMETRIC DETECTION OF PYOCYANIN IN NANOFLUIDIC CHANNELS

Microfabricated nanofluidic electrode assemblies (NEAs) with integrated palladium references were used to amperometrically monitor changes in pyocyanin concentration. Pyocyanin is an electroactive molecule that is produced by the opportunistic pathogen Pseudomonas aeruginosa and is directly linked to cellular processes that increase both robustness and virulence in this bacterium. This is the first time that pyocyanin has been measured in real time using microfabricated sensors. A linear response in faradaic current (R2 = 0.96) was observed over a biomedically relevant range of pyocyanin concentrations (0–100 μM) while continuously measuring the current for 2 h. Measurement of the current that results from the repeated oxidation and reduction of pyocyanin at two closely spaced electrodes inside the device nanochannel yielded a 1.07 μM limit of detection without electrical isolation of the electrochemical cell. Since a reference electrode is integrated inside the nanofluidic channel of these sensors, they can potentially be employed to detect pyocyanin and other redox-active molecules in wide range of medical and environmental settings where space is limited. NEAs were also used with an external Ag/AgCl reference electrode to determine the concentration of pyocyanin in trypticase soy broth samples. This type of analysis is completed in less than 2 min and the detection limit was determined to be 441 nM.

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.

Improved monitoring of P. aeruginosa on agar plates

Described is the fabrication of a disposable electrochemical assay that is integrated with standard Kings A agar culture plates, for the selective and specific detection of Pseudomonas aeruginosa. Agar plates provide several advantages over liquid culture, including protecting the sensor from biofouling and faster identification in small sample volumes. Cultures of P. aeruginosa, starting from initial cell counts of 102 to 108 cells in 5 microliter volumes, were incubated at 23, 37, and 42 °C and monitored both visually and electrochemically. Square wave voltammetry scans confirmed the production of a redox species, pyocyanin, over time that was dependent on the initial load of cells. The pyocyanin easily diffuses through the agar to reach the electrode surface. Using this simple and cheap approach, positive identification of P. aeruginosa was achieved several hours faster via electrochemical detection compared to traditional visual analysis.

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 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.

Monitoring Pseudomonas aeruginosa in culture plates using embedded electrochemical sensors

Described is the fabrication of a disposable electrochemical assay, embedded in solid growth media agar. By applying a unique set of potentials and measuring current change using electrochemical sensors, we were able to selectively detect the presence of Pseudomonas aeruginosa. Bacterial cultures of P. aeruginosa, Escherichia coli, and Staphylococcus aureus were grown on these plates for a period of 48 hours at room temperature. The maximum currents from square-wave voltammetric scans showed the production of redox active molecules by P. aeruginosa after 15 hours of growth. This simple disposable electrochemical approach for detecting P. aeruginosa could have implications for diagnosing the presence of P. aeruginosa infections in limited resource areas.