Discriminating Typical and Atypical Cystic Fibrosis-Related Bacteria by Multiplex PNA-FISH

Abstract

This study aims to report the development of peptidenucleic acid (PNA) probes to specifically detect the cystic fibrosis(CF)-associated traditional and atypical species Pseudomonasaeruginosa and Inquilinus limosus, respectively. PNA probes weredesigned in silico, developed and tested in smears prepared inphosphate-buffer saline (PBS), and in artificial sputum medium(ASM). A multiplex fluorescent in situ hybridization (FISH)approach using the designed probes was further validated inartificially contaminated clinical sputum samples and also appliedin polymicrobial 24 h-old biofilms involving P. aeruginosa,I. limosus, and other CF-related bacteria. Both probes showedhigh predictive and experimental specificities and sensitivities. Themultiplex PNA-FISH assay, associated with non-specific staining,was successfully adapted in the clinical samples and in biofilms ofCF-related bacteria, allowing differentiating the communitymembers and inferring about microbial-microbial interactionswithin the consortia. This study revealed the great potential of PNAFISH as a diagnostic tool to discriminate between classical and lesscommon CF-associated bacteria, being suitable to further describespecies-dependent prevention strategies and deliver more effectivetarget control therapeutics.