Changes In Subgingival Microbial Profiles In Adult Periodontitis Subjects Received Non-surgical Periodontal Treatment

OBJECTIVETo observe changes in subgingival microbial profiles in adult periodontitis subjects received non - surgical periodontal treatment and find out putative periodontal pathogens and aid the clinical plan.METHODS1. Subject and Population26 adult periodontitis subjects 30 - 55 years of age who had not received previous periodontal therapy were selected for study. Subjects came from the Stomatology College of China Medical University and had at least 8 pockets at posterior teeth with pocket depth between 3 -6mm. Subjects were excluded from the study if they had used any antibiotics in the latest 2 months or had any systemic condition which could affect the progress of periodontitis.2. Clinical measurement and samplingA site with pocket depth of 3 - 6mm in each subject was selected. At the site the plaque index ( PI I) , bleeding on probing ( BOP) , shakiness, radiographic bone loss and probing depth (PD) were recorded at baseline, 7, 30, 90 days after therapy. Subgingival plaque samples were taken every time. After baseline monitoring all subjects received full mouth scaling and root planning (SRP), systemically administered metronidazole (200mg, TID) for 7 days.3. DNA extractionGenoraic DNA was separated from plaque samples and the concentration of DNA was calculated spectrophotometrically by measurement of absorption at 260 nm, and the quality was estimated by theA260/A280 rati?4. Polymerase chain reactionThe extracted DNA was amplified with primers taking advantage of 16S rDNA. Primers with " GC" clamp were designed to perform the " Y" structure in the later DGGE.5. DGGEThe full bacteria DNA were separated through DGGE. Two bands were selected which were intensive at baseline and disappeared after therapies. In addition, two bands newfound were recruited. Then DGGE bands were sequenced after excision from the gel and reamplifi-cation.RESULTS1. Changes in clinical parameters over timeThere were significant decrease in PI I, BOP, shakiness and PD (p <0. 01). The changes in radiographic bone loss were not statistically significant.2. Regression analysis showed that the change in BOP was significantly and positively related to the change in PD ( p < 0. 01, r = 0. 710) , while bone loss and PD were moderately related to shakiness (p < 0.01, r = 0.630 and 0.526 respectively).3. Effect of treatment on the concentration of DNAAt the site basis the concentration of DNA was decreased from39.44( +23. 56) ug/ml to 24. 68( + 10. 22) ug/ml, But it was not significant.4. Fingerprinting of subgingival microbial DNA in DGGEWe got a subgingival microbial DNA fingerprinting in the DGGE. It showed that there was a remarkable change that new bands came into being at the 30 days. While the DNA fingerprinting of 90 days was somewhat similar to that of baseline.5. Identifying and sequencingWe sequenced 4 DNA bands including 2 bands disappeared and 2 bands newfound after therapy. The sequences retrieved were determined by a BLAST search in GenBank. The disappeared 2 bands showed 98% and 99% similarity with 16S rDNA fragments of Pg respectively. The newfound bands had 99% similarity with Porphyromonas sp . oral clone cw034.CONCLUSION1. Non - surgical periodontal treatment might significantly improved clinical parameters such as P1I, PD, BOP and shakiness. At the site basis the concentration of subgingival microbial DNA was decreased , but it was not significant. The proportion between periodontal pathogens and compatible microbial was changed by therapies in dental plaque samples.2. Regression analysis showed that the change in BOP was significantly and positively related to the change inPD ( p<0.01, r = 0.710); In addition shakiness and PD were moderately related to bone loss ( p<0.01, r= 0.630 and 0.526 respectively).3. Pg was a major putative pathogen in AP, frequently it took animportant role in the etiology of periodontitis.4. The use of PCR and DGGE gave us a reliable fingerprinting of subgiginval microbial...