Author(s):
Mendes, Luzia ; Rocha, Rui ; Azevedo, Andreia S. ; Ferreira, Catarina ; Henriques, Mariana ; Pinto, Miguel Gonçalves ; Azevedo, Nuno Filipe
Date: 2016
Persistent ID: https://hdl.handle.net/1822/44858
Origin: RepositóriUM - Universidade do Minho
Project/scholarship:
info:eu-repo/grantAgreement/FCT/5646-ICCMS/82815/PT
;
info:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBDE%2F51910%2F2012/PT;
info:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F82663%2F2011/PT;
Subject(s): PNA-FISH; Tissue invasion; Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis; Periodontal disease; Science & Technology
Description
Purpose: We aim to develop peptic nucleic acid (PNA) probes for the identification and localization of Aggregatibacter 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 to allow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex). After being tested on representative strains of P. gingivalis and A. actinomycetemcomitans, the PNA-FISH method was then adapted to detect microorganisms in the subgingival plaque and gingival samples, collected from patients with severe periodontitis. Results The best hybridization conditions were found to be 59 °C for 150 min for both probes (PgPNA1007 and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and 99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH method was able to detect and discriminate target bacteria in the mixed microbial population of the subgingival plaque 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 technique we were able to observe spatial distribution of these species within polymicrobial communities in the periodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.
The authors report no conflicts of interest. This work was financially supported by: Project POCI-01-0145-FEDER-006939 Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI)—and by national funds through FCT—Fundação para a Ciência e a Tecnologia; PIC/IC/82815/2007, SFRH/BDE/51910/2012, SFRH/BD/82663/2011].
