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Published on: October 14, 2011
Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye
Julia E Egido1, Catherine Toner-Bartelds1, Ana Rita Costa2,3,4
1Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Abstract:
Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.
Insights
We developed a rapid, fluorescence-based method to select effective bacteriophages (phages) for treating bacterial infections. This technique visualizes phage infection dynamics in real time, aiding therapeutic development.
Area of Science:
- Microbiology
- Virology
- Biotechnology
Background:
- Bacteriophages (phages) are viruses that infect bacteria and are a promising alternative to antibiotics.
- Selecting effective lytic phages requires robust methods to monitor infection dynamics.
- Current phage selection methods are time-consuming and lack real-time monitoring capabilities.
Purpose of the Study:
- To develop a high-throughput, real-time method for selecting effective bacteriophages.
- To enable visualization of phage infection dynamics for therapeutic applications.
- To provide a reliable tool for optimizing phage selection against Gram-negative bacteria.
Main Methods:
- Utilized a membrane-impermeant nucleic acid dye to detect lysed bacteria and phage progeny.
- Monitored phage infection in real time via fluorescence signal readout.
- Validated the method on Pseudomonas aeruginosa and Klebsiella pneumoniae, comparing results with traditional techniques.
Main Results:
- Demonstrated that increased fluorescence correlates with phage-mediated bacterial killing.
- Confirmed the method's efficacy through spot tests, colony plating, flow cytometry, and metabolic activity assays.
- Showcased the method's utility in comparing phage activity and screening clinical isolates.
Conclusions:
- The developed fluorescence-based method offers a fast and reliable approach for selecting bacteriophages.
- This technique facilitates real-time monitoring of phage infection dynamics, crucial for therapeutic development.
- The method is valuable for optimizing the selection of phages against Gram-negative pathogens.
