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'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens
Published on: July 8, 2011
Rapid phage susceptibility determination using a semi-automated bioluminescent ATP assay
Houssein Chalhoub1,2, Valéry Daubie2,3, Hafid Dahma2,4
1Center for Environmental and Occupational Health, Ecole de Santé Publique - Université Libre de Bruxelles, Brussels, Belgium.
Abstract:
Antimicrobial resistance is a major threat to human health. Phage therapy is a promising alternative to antibiotics, yet routine diagnostic tools capable of rapidly determining phage susceptibility are lacking. Our goal was to develop a diagnostic tool providing easy-to-interpret in vitro phage activity results within a clinically relevant time frame. We developed a prototype based on bioluminescence emitted by adenosine triphosphate (ATP) release during phage-mediated bacterial lysis. Ten phages were tested against 20 clinical isolates of four species: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. Overall concordance with double agar overlay (DAO) was fair (72%). The prototype showed high sensitivity (94.5%) at detecting weak killers and high specificity (87%) at identifying strong killer phages. Most discordances involved DAO weak killers classified as active by the prototype. Most of these discordant phage-bacteria couples produced a spot in the spot test, suggesting the assay may detect but not distinguish killing without replication from killing with replication. The method showed high repeatability and reproducibility. A few aberrant species specificity results highlighted the need for routine controls. Results from prospective urine samples were obtained within an average of 5 h.
Importance:
The lack of reproducible user-friendly phage susceptibility testing that can timely inform clinical decisions remains a major barrier to the routine clinical use of phages. We developed a high-throughput, semi-automated prototype that delivers easy-to-interpret phage susceptibility results for four bacterial species within 5 h in a "real-life" clinical flow. However, the correlation between in vitro lytic activity and clinical outcomes still needs to be established to meet regulatory requirements for in vitro diagnostic (IVD) medical devices.
Insights
A new diagnostic tool uses bioluminescence to rapidly assess bacterial susceptibility to bacteriophages (phages), offering a promising alternative to antibiotics. This rapid phage susceptibility testing provides results within 5 hours for clinical use.
Area of Science:
- Microbiology and Infectious Diseases
- Biotechnology and Biomedical Engineering
Background:
- Antimicrobial resistance (AMR) poses a significant global health threat, necessitating novel therapeutic strategies.
- Bacteriophage (phage) therapy presents a viable alternative to conventional antibiotics, but its clinical application is hindered by the lack of rapid susceptibility testing.
- Existing phage susceptibility assays are often time-consuming and not user-friendly, delaying clinical decision-making.
Purpose of the Study:
- To develop and evaluate a novel, high-throughput, semi-automated diagnostic prototype for rapid phage susceptibility testing.
- To provide easy-to-interpret in vitro phage activity results within a clinically relevant timeframe (under 5 hours).
- To assess the performance of the prototype against established methods like the double agar overlay (DAO) assay.
Main Methods:
- A bioluminescence-based prototype assay was developed, detecting adenosine triphosphate (ATP) release indicative of phage-mediated bacterial lysis.
- Ten phages were tested against 20 clinical isolates from four key bacterial species: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae.
- The prototype's results were compared with the double agar overlay (DAO) method for concordance, sensitivity, and specificity.
Main Results:
- The prototype demonstrated fair overall concordance (72%) with the DAO method.
- High sensitivity (94.5%) was achieved in detecting weak phage killers, and high specificity (87%) in identifying strong killer phages.
- Results from prospective urine samples were obtained within an average of 5 hours, demonstrating clinical applicability. The assay showed high repeatability and reproducibility.
Conclusions:
- The developed bioluminescence-based prototype offers a rapid, user-friendly, and reproducible method for assessing phage susceptibility.
- The assay shows potential for routine clinical use, providing timely results to guide phage therapy decisions.
- Further studies are needed to establish the correlation between in vitro lytic activity and clinical outcomes for regulatory approval as an in vitro diagnostic (IVD) device.
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