Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Biofouling
Diana Vilas BoasNuno F Azevedo

Abstract

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

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Citations

Oct 2, 2020·Annals of Clinical Microbiology and Antimicrobials·Zahra CheginiAref Shariati
Oct 12, 2017·Frontiers in Cellular and Infection Microbiology·Stephanie A FongPeter-John Wormald
May 16, 2019·International Forum of Allergy & Rhinology·Michelle Menon Miyake, Benjamin S Bleier
May 28, 2020·Antibiotics·Celia Ferriol-González, Pilar Domingo-Calap
Nov 1, 2017·Nature Microbiology·Lucia VidakovicKnut Drescher
Mar 14, 2021·Current Opinion in Biotechnology·Joana AzeredoZuzanna Drulis-Kawa
Jun 30, 2021·Annual Review of Virology·Diana P PiresJoana Azeredo

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Datasets Mentioned

BETA
KT192572
AY078382

Methods Mentioned

BETA
flow cytometry

Software Mentioned

ClustalW
FV10
RNA Chemistry Laboratory
Probe Match
Blast
- ASW
phageFISH
Blastn

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