title: Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).
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.
authors.affiliation: b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEPABE), Department of Chemical Engineering, Faculty of Engineering , University of Porto , Porto , Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Vilas Boas
authors.affiliation: a LIBRO -Laboratório de Investigação em Biofilmes Rosário Oliveira , Centre of Biological Engineering, University of Minho , Braga , Portugal.|b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEPABE), Department of Chemical Engineering, Faculty of Engineering , University of Porto , Porto , Portugal.
authors.forename: Carina
authors.initials: C
authors.lastname: Almeida
authors.affiliation: a LIBRO -Laboratório de Investigação em Biofilmes Rosário Oliveira , Centre of Biological Engineering, University of Minho , Braga , Portugal.
authors.forename: Sanna
authors.initials: S
authors.lastname: Sillankorva
authors.affiliation: b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEPABE), Department of Chemical Engineering, Faculty of Engineering , University of Porto , Porto , Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Nicolau
authors.affiliation: b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEPABE), Department of Chemical Engineering, Faculty of Engineering , University of Porto , Porto , Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Azeredo
authors.affiliation: b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEPABE), Department of Chemical Engineering, Faculty of Engineering , University of Porto , Porto , Portugal.
authors.forename: Nuno F
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D040981:Acinetobacter baumannii / Q000821:virology*; D001435:Bacteriophages / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009841:Oligonucleotides / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000821:virology*; D015203:Reproducibility of Results
chemical_list: D009841:Oligonucleotides; C477371:locked nucleic acid
keywords: Acinetobacter; Pseudomonas aeruginosa; bacteriophage; biofilms; phage infection, LNA-FISH