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Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are...

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Autores principales: Melo, Luís D. R., Monteiro, Rodrigo, Pires, Diana P., Azeredo, Joana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8868278/
https://www.ncbi.nlm.nih.gov/pubmed/35203767
http://dx.doi.org/10.3390/antibiotics11020164
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author Melo, Luís D. R.
Monteiro, Rodrigo
Pires, Diana P.
Azeredo, Joana
author_facet Melo, Luís D. R.
Monteiro, Rodrigo
Pires, Diana P.
Azeredo, Joana
author_sort Melo, Luís D. R.
collection PubMed
description Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.
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spelling pubmed-88682782022-02-25 Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages Melo, Luís D. R. Monteiro, Rodrigo Pires, Diana P. Azeredo, Joana Antibiotics (Basel) Article Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice. MDPI 2022-01-27 /pmc/articles/PMC8868278/ /pubmed/35203767 http://dx.doi.org/10.3390/antibiotics11020164 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Melo, Luís D. R.
Monteiro, Rodrigo
Pires, Diana P.
Azeredo, Joana
Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title_full Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title_fullStr Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title_full_unstemmed Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title_short Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages
title_sort phage-host interaction analysis by flow cytometry allows for rapid and efficient screening of phages
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8868278/
https://www.ncbi.nlm.nih.gov/pubmed/35203767
http://dx.doi.org/10.3390/antibiotics11020164
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