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

Liliam K. Harada,José M. Oliveira Júnior,Matthieu Tubino,Victor M. Balcão,Waldemar Bonventi Júnior,Fernanda C. Moreli,Erica C. Silva,Thais J. Oliveira,Marta M. D. C. Vila

doi:10.3390/bios11040124

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.

Highlights

Among the microorganisms that cause hospital infections one can highlight the multidrug-resistant Pseudomonas aeruginosa, which represents a major threat to patients in health care services [1,2,3,4]
For bacterial cell numbers up to 107, no visible change could be observed beyond 180 min of assay (Figure 10), allowing us to infer that the presence of this bacterium in a fluid could be detected within a 3 h timeframe assay
We propose two bio-detection systems for P. aeruginosa, based on a cocktail of three different lytic phages isolated from environmental sources in Brazil, which were structurally and functionally stabilized within a biopolymeric matrix

Summary

Introduction

Among the microorganisms that cause hospital infections one can highlight the multidrug-resistant Pseudomonas aeruginosa, which represents a major threat to patients in health care services [1,2,3,4]. The surge of multiple bacterial resistance to conventional antibiotics [5,6] has been gaining increasing worldwide awareness, leading to a renewed interest in phage particles by the scientific community, with these entities (devoid of any metabolic machinery) being re-discovered as having high-potential in biopharmaceutical applications, such as antimicrobial or bacterial biosensing [1,2,7,8,9,10,11,12]. P. aeruginosa has evolved over time, becoming progressively more resistant to the available antibiotics, and is considered one of the six most dangerous pathogens responsible for hospital-acquired infections [19,20,21]

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Results

Discussion

Conclusion