Print Email Facebook Twitter Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye Title Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye Author Egido, Julia E. (University Medical Center Utrecht) Toner-Bartelds, Catherine (University Medical Center Utrecht) Martins Costa, A.R. (TU Delft BN/Stan Brouns Lab; Kavli institute of nanoscience Delft; Fagenbank) Brouns, S.J.J. (TU Delft BN/Stan Brouns Lab; Kavli institute of nanoscience Delft; Fagenbank) Rooijakkers, Suzan H.M. (University Medical Center Utrecht) Bardoel, Bart W. (University Medical Center Utrecht) Haas, Pieter Jan (University Medical Center Utrecht) Date 2023 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. To reference this document use: http://resolver.tudelft.nl/uuid:2ffe569b-779d-4058-8167-0182a80bc4aa DOI https://doi.org/10.1038/s41598-023-27734-w ISSN 2045-2322 Source Scientific Reports, 13 (1) Part of collection Institutional Repository Document type journal article Rights © 2023 Julia E. Egido, Catherine Toner-Bartelds, A.R. Martins Costa, S.J.J. Brouns, Suzan H.M. Rooijakkers, Bart W. Bardoel, Pieter Jan Haas Files PDF s41598_023_27734_w.pdf 1.39 MB Close viewer /islandora/object/uuid:2ffe569b-779d-4058-8167-0182a80bc4aa/datastream/OBJ/view