title: Discriminating typical and atypical cystic fibrosis-related bacteria by multiplex PNA-FISH.
abstract: This study aims to report the development of peptide nucleic acid (PNA) probes to specifically detect the cystic fibrosis (CF)-associated traditional and atypical species Pseudomonas aeruginosa and Inquilinus limosus, respectively. PNA probes were designed in silico, developed and tested in smears prepared in phosphate-buffer saline (PBS), and in artificial sputum medium (ASM). A multiplex fluorescent in situ hybridization (FISH) approach using the designed probes was further validated in artificially contaminated clinical sputum samples and also applied in polymicrobial 24 h-old biofilms involving P. aeruginosa, I. limosus, and other CF-related bacteria. Both probes showed high predictive and experimental specificities and sensitivities. The multiplex PNA-FISH assay, associated with non-specific staining, was successfully adapted in the clinical samples and in biofilms of CF-related bacteria, allowing differentiating the community members and inferring about microbial-microbial interactions within the consortia. This study revealed the great potential of PNA-FISH as a diagnostic tool to discriminate between classical and less common CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics. Biotechnol. Bioeng. 2017;114: 355-367. © 2016 Wiley Periodicals, Inc.
authors: Centre of Biological Engineering, LIBRO-Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Susana P
authors: SP
authors: Lopes
authors: LEPABE, Faculty of Engineering, Department of Chemical Engineering, University of Porto, Porto, Portugal.
authors: Daniel T
authors: DT
authors: Carvalho
authors: Centre of Biological Engineering, LIBRO-Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors: Maria O
authors: MO
authors: Pereira
authors: LEPABE, Faculty of Engineering, Department of Chemical Engineering, University of Porto, Porto, Portugal.
authors: Nuno F
authors: NF
authors: Azevedo
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015373:Bacterial Typing Techniques; D018441:Biofilms; D003550:Cystic Fibrosis / Q000382:microbiology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D064307:Microbiota; D020135:Peptide Nucleic Acids / Q000032:analysis* / Q000378:metabolism; D013183:Sputum / Q000382:microbiology
chemical_list: D020135:Peptide Nucleic Acids
keywords: Inquilinus limosus; PNA probe; Pseudomonas aeruginosa; cystic fibrosis microbiome; fluorescence in situ hybridization