Electrochemical detection of Pseudomonas aeruginosa in human fluid samples via pyocyanin

Webster, Thaddaeus A.; Sismaet, Hunter J.; Conte, Jared L.; Chan, I-ping J.; Goluch, Edgar D.

doi:10.1016/j.bios.2014.04.028

Cited by 99 publications

(94 citation statements)

References 33 publications

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“…2A) important for P. aeruginosa virulence. Because it is under strict QS control (48) and can be detected electrochemically (45,52,54,55), pyocyanin can be used as a proxy for QS-mediated communication. To determine the number of bacteria required for P. aeruginosa to initiate QS, 8-pL microtraps were printed directly around one to five P. aeruginosa cells in situ (28), and the pyocyanin concentration was monitored by SECM imaging as the population grew over time.…”

Section: Resultsmentioning

confidence: 99%

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy

Connell

Kim

Shear

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

166

162

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Microbes frequently live in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.Pseudomonas aeruginosa | scanning electrochemical microscopy | quorum sensing | 3D printing | pyocyanin B acterial populations are often found in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells (1-5). These aggregates serve as building blocks for larger biofilm communities as well as a primary mode of transmission for pathogenic microbes (5-8). Similar to biofilm communities, aggregates develop microscale physical and chemical heterogeneity and display clinically relevant phenotypes, including enhanced antibiotic resistance (2,(8)(9)(10)(11)(12)(13)(14)(15)(16). Moreover, aggregate sizes containing as few as 10 3 bacteria have been shown to engage in quorum sensing (QS)-mediated behaviors (17-21). In its simplest form, QS is a communication strategy that allows bacteria to effectively monitor their population density through the secretion and sensing of extracellular signals (7,(22)(23)(24). When the population reaches a specific density, activation of the QS regulatory cascade results in enhanced transcription of a defined set of genes. These genes control distinct behaviors, including virulence, in the opportunistic pathogen Pseudomonas aeruginosa (25). In addition to displaying QS-mediated behaviors, bacterial aggregates have been shown to interact with neighboring aggregates both in vitro and in vivo (9,(26)(27)(28). Indeed, these interactions have a profound impact on virulence and are often mediated by small diffusible molecules (8-10, 22, 29-31).Despite the prevalence of aggregates in nature, understanding the mechanisms controlling their behavior and intera...

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Section: Resultsmentioning

confidence: 99%

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy

Connell

Kim

Shear

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

166

162

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“…8 Agar plates can also be used with serial dilutions of antibiotics to determine the minimum inhibitory concentration (MIC) required to kill the bacteria. 16 PYO detection is oen performed via solvent extraction to remove the molecule of interest from cell cultures (liquid and agar) and then measured optically or electrochemically. 10 In some instances, a second form of identication is required aer incubation.…”

Section: Introductionmentioning

confidence: 99%

“…11 A more modern approach is the use of polymerase chain reaction (PCR) schemes to increase the amount of pathogenic DNA or RNA present in a sample, which is then used for rapid identication. Several groups have identied P. aeruginosa in liquid cultures by detecting pyocyanin electrochemically, 16,[19][20][21][22] but this format is not typically employed by pathologists for analysis. 13,14 PCR-based approaches are much more selective and sensitive; however, they use expensive reagents and require specialized personnel or expensive instrumentation to handle the sample prep and data analysis.…”

Section: Introductionmentioning

confidence: 99%

Improved monitoring of P. aeruginosa on agar plates

et al. 2015

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Described is the fabrication of a disposable electrochemical assay that is integrated with standard King's 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 10 2 to 10 8 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. † Electronic supplementary information (ESI) available: ESI contains a calibration curve for PYO measured in TSB media, solid TSB agar, and solid King's A agar; a plot of the measured maximum current vs. time for the bacterial loads used in Fig. 4; curves from which the maximum PYO production rate in Fig. 2B was calculated; and scanning electron microscopy images of P. aeruginosa grown on King's A agar as well as the surface of the electrodes aer cell growth. See

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“…Pyocyanin oxidizes, like all phenazines, at a negative voltage, −247 mV (Bellin et al, 2014; Webster et al, 2014), which is visible as a peak in the current at −240 mV in Figures 4C,D. The reduction of pyocyanin occurs at −300 mV (Bellin et al, 2014), which is visible by the inverse peak at −280 mV.…”

Section: Methodsmentioning

confidence: 99%

“…Pyocyanin on the other hand oxidizes at a negative voltage (−247 mV). This means that, at these voltages only phenazines will be detected (Webster et al, 2014). Pyocyanin is therefore an interesting candidate for use as a bacterial output product because there is only a small chance of a false positive signal with other products.…”

Section: Introductionmentioning

confidence: 99%

Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay

Dierckx

Puyvelde

Venken

et al. 2015

Front. Bioeng. Biotechnol.

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IntroductionAuxins are hormones that regulate plant growth and development. To accurately quantify the low levels of auxins present in plant and soil samples, sensitive detection methods are needed. In this study, the design and construction of two different whole cell auxin bioassays is illustrated. Both use the auxin responsive element HpaA as an input module but differ in output module. The first bioassay incorporates the gfp gene to produce a fluorescent bioassay. Whereas the second one utilizes the genes phzM and phzS to produce a pyocyanin producing bioassay whose product can be measured electrochemically.ResultsThe fluorescent bioassay is able to detect 4-hydroxyphenylacetic acid (4-HPA) and 2-phenylacetic acid (PAA) concentrations from 60 μM to 3 mM in a dose-responsive manner. The pyocyanin producing bioassay can detect 4-HPA concentrations from 1.9 to 15.625 μM and PAA concentrations from 15.625 to 125 μM, both in a dose-responsive manner.ConclusionA fluorescent whole cell auxin bioassay and an electrochemical whole cell auxin bioassay were constructed and tested. Both are able to detect 4-HPA and PAA at concentrations that are environmentally relevant to plant growth.

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Electrochemical detection of Pseudomonas aeruginosa in human fluid samples via pyocyanin

Cited by 99 publications

References 33 publications

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy

Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy

Improved monitoring of P. aeruginosa on agar plates

Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay

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