Avaliação das relações de prematuridade e doença periodontal através da detecção de DNA genômico de Porphyromonas gingivalis e Aggregatibacter actinomycetemcomitans em biofilme bucal materno e tecido placentário

Abstract

Periodontal disease includes a number of changes that occur in the periodontium from the formation of biofilm, characterized by the presence of a range of microrganisms. Some of these, by their unique characteristics, are strongly associated with disorders such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The aim of this study was to evaluate the relationship of preterm birth and periodontal disease by detecting genomic DNA of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in oral biofilm maternal and placental tissue. For these purposes, oligonucleotide primers were designed for the detection of RNA polymerase gene of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans leukotoxin as targets for PCRtr amplification from moutht exudate biofilm of the gingival sulcus, placenta and umbilical cord blood, 19 women experiencing preterm labor. Also held a screening for PCRtr 64 archived samples of genomic DNA from placentas of mothers of infants with suspected neonatal infection. As normalization genes, it was used B-actin, for DNA samples and S26 for RNA. To confirm and evaluate both presence and bacterial viability, RT-PCRtr was applied to determine mRNA expression of RNA polymerase from Porphyromonas gingivalis and leukotoxin from Aggregatibacter actinomycetemcomitans, in total RNA from placenta. Additionally, confocal microscopy of placenta tissues PCRtr positives, were also investigated. The results demonstrated that primers were efficients and specific for the diagnosis and expression studies and all specimens studied were viable for the detection of genomic DNA of microrganisms investigated. The PCRtr detected Agregatibacter actinomycetemcomitans , around 15%, in the three specimens, however, the biofilm samples of the mothers had the highest percentage of 16.7%. A periodontal evaluation conducted found that 50% of women had bleeding gums, calculating 83% and 17% periodontal pocket. Prevalence of Porphyromonas gingivalis was elevated in the specimens studied, 100% in umbilical cord blood, 95% in the placental hilus and 83% of biofilms of maternal gingival sulcus. PCRtr screening of 64 archived samples of placental genomic DNA, reinforced the findings of prevalence of Porphyromonas gingivalis, demonstrating 32 (50%) positive and 2 (3.1%) positive for Aggregatibacter actinomycetemcomitans. RT-PCRtr of total RNA from placenta presented 5 (31%) samples with Leukotoxin mRNA expression of Agregatibacter actinomycetemcomitans and 12 (75%) showed the expression of the RNA polymerase specific for the Porphyromonas gingivalis. These findings show that in the PCRtr positive placentas a large percentage of viable bacteria are expressing relevant proteins for their proliferation. The confocal microscopy data reinforce these findings, suggesting the presence of bacterial genetic material by consistent formation of vacuoles, significant bacteria and extra-nuclear genetic material. The results obtained from the different methodologies applied in specimens evaluated, demonstrate a significant relation of prematurity with maternal periodontal disease.