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 technicallimitations of traditional methods. The aim of this study was to provide a direct fluorescence in situhybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phagereplication phase, allowing the study of population dynamics during infection. Bacteriophagesspecific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected.Four LNA probes were designed and optimized for phage-specific detection and for bacterialcounterstaining. To validate the method, LNA-FISH counts were compared with the traditionalplaque 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) wasobserved between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a gooddiscrimination of the infected cells and also allowed the assessment of the spatial distribution ofinfected and non-infected populations.