Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Abstract

Purpose: We aim to develop peptic nucleic acid (PNA) probes for the identification and localization ofAggregatibacter actinomycetemcomintans and Porphyromonas gingivalis in sub-gingival plaque and gingival biopsies by Fluorescence in situ Hybridization (FISH).Methods: A PNA probe was designed for each microorganism. The PNA-FISH method was optimized toallow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex). Afterbeing tested on representative strains of P. gingivalis and A. actinomycetemcomitans,the PNA-FISH methodwas then adapted to detect microorganisms in the subgingival plaque and gingival samples, collectedfrom patients with severe periodontitis.Results: The best hybridization conditions were found to be 59 ◦C for 150 min for both probes (PgPNA1007and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH methodwas able to detect and discriminate target bacteria in the mixed microbial population of the subgingivalplaque and within periodontal tissues.Conclusion: This investigation presents a new highly accurate method for P. gingivalis and A. actinomycetemcomitans detection and co-location in clinical samples, in just few hours. With this techniquewe were able to observe spatial distribution of these species within polymicrobial communities in theperiodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.