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. 2021 Apr 15;11(4):124.
doi: 10.3390/bios11040124.

Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains

Affiliations

Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains

Liliam K Harada et al. Biosensors (Basel). .

Abstract

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet's biosphere, killing between 4-50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13-15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

Keywords: Pseudomonas aeruginosa; bacterial biosensing; bacteriophage particles; bio-reactive polymeric matrix; chromogenic/bioluminescent bio-hydrogel; immobilization and structural/functional stabilization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bio-detection system I, consisting of a six-well culture plate within which the chromogenic formulations integrating a cocktail of phage particles and sodium 1,2-naftoquinone-4-sulfonate together with specific amounts of gelatin and casein were poured and allowed to polymerize.
Figure 2
Figure 2
Bio-detection system II, consisting of a small cylindrical equipment machined in rigid white PVC, having approximately 6 cm in height and 6 cm in diameter, divided into a lower part (A) where the bio-reactive hydrogel integrating the immobilized phage cocktail, luciferin, luciferase, ADP (adenosine 5′-diphosphate sodium salt), and Mg2+ is housed, and an upper part (B) housing the light sensors, which was used to cover (A). The heart of the system designed consisted of a receptacle for the biopolymeric matrix containing the phage cocktail, above which the light sensors were attached and connected to an Arduino platform, which, in turn, was connected via a USB port to a notebook computer.
Figure 3
Figure 3
Results obtained following evaluation of the lytic activity of the isolated phage particles ph0031, ph0034, and ph0041, and a comparison with the lytic activity of phage JG004 obtained from the DSMZ collection (a) and of the lytic activity of the phage cocktail produced with the three isolated phage particles (b) on a P. aeruginosa DSM19880 bacterial lawn.
Figure 4
Figure 4
Results from evaluation of the lytic activity of the immobilized phages following integration within the bio-reactive polymeric matrix with concomitant structural and functional stabilization. (a) Bio-reactive polymeric matrix devoid of phage particles. (b) Bio-reactive polymeric matrix integrating the cocktail of phage particles.
Figure 5
Figure 5
DESEM (dispersive-energy scanning electron microscopy) photomicrographs of the bioluminescent/chromogenic hydrogel surface ((a) ×50, (b) ×250, and of the cross-section fracture zone (c) ×250).
Figure 6
Figure 6
Images obtained by X-ray tomographic analyses of the bioluminescent/chromogenic hydrogel, being (a) a leaning surface view allowing us to observe the top side (right thicker surface in green), and (b) a leaning surface view allowing us to observe the bottom side (left thin surface in green).
Figure 7
Figure 7
Results obtained in the optimization of the amounts of phage particles, sodium 1,2-naftoquinone-4-sulfonate, gelatin, and casein, leading to the bio-reactive chromogel for bio-detection system I, displaying the time evolution of the color produced using a fixed (added) amount of P. aeruginosa cells. The composition of formulations 1, 2, and 3 is displayed in Table 1.
Figure 8
Figure 8
Normalized integrated color density of the three chromogels containing phage particles, sodium 1,2-naftoquinone-4-sulfonate, gelatin, and casein, further contacted with a fixed amount of P. aeruginosa cells, throughout the bioreaction time, allowing us to select an optimized bio-reactive chromogel. Values represent the mean of three independent assays. Error bars represent the standard deviation.
Figure 9
Figure 9
Results obtained using the selected bio-reactive chromogel with optimized concentrations of phage particles, sodium 1,2-naftoquinone-4-sulfonate, gelatin, and casein, in bio-detection system II, with evolution throughout the time of the color produced using variable added amounts of P. aeruginosa cells. As a control for the colorimetric bioreaction, cells of S. aureus CCCD-S009 were also contacted with the selected bio-reactive chromogel (Figure 9, bottom right).
Figure 10
Figure 10
Normalized integrated color density of the optimized bio-reactive chromogel integrating phage particles, sodium 1,2-naftoquinone-4-sulfonate, gelatin, and casein, further added with variable amounts of P. aeruginosa cells, throughout the bioreaction time. Cells of S. aureus CCCD-S009 were used as a control for the bio-detection assays. Values represent the mean of three independent assays. Error bars represent the standard deviation.
Figure 11
Figure 11
Receptacle for the bio-reactive hydrogel in bio-detection system II, showing the position of the two light sensors (a), macroscopic aspect of the freshly-prepared phage-containing bio-reactive hydrogel (b), and macroscopic aspect of the phage-containing bio-reactive hydrogel with produced bioluminescence following exposure to P. aeruginosa cells (c).
Figure 12
Figure 12
Evolution of the normalized LDR (Light-Dependent Resistor) signal throughout the bioreaction timeframe, using the bioluminescent hydrogel in bio-detection system II.
Figure 13
Figure 13
Evolution of the normalized solid-state photosensor signal throughout the bioreaction timeframe using the bioluminescent hydrogel in bio-detection system II.

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