Discriminating between typical and atypical cystic fibrosis-related bacterial species 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 and in artificial sputum medium. A multiplex fluorescent in situ hybridization (FISH) approach using the probes was further validated in artificially contaminated clinical sputum samples and also applied to polymicrobial 24h-old biofilms involving the aforementioned and other CF-related bacteria. Both probes showed high predictive and experimental specificities/sensitivities. The multiplex PNA-FISH, 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 emerging CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics.