Computational protocol: Partial denture metal framework may harbor potentially pathogenic bacteria

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Protocol publication

[…] The dental records of 130 partially dentate patients referred to the Piracicaba School of Dentistry, State University of Campinas, Sao Paulo, Brazil, were assessed. Individuals were eligible if they were completely edentulous at the maxilla and used previous complete denture and partially edentate at the mandible with no previous RPD (n=54). Twenty eight individuals did not respond to the telephone contact. The remaining (n=26) were clinically examined and included if they were systemically healthy, without periodontitis (clinical attachment loss no more than 3 mm with no bleeding on probing), no smokers, with no presence of restorations, active caries, and crowns, no need of pre-prosthetic surgery, no prescription of systemic antibiotics or chemical plaque control in the last six months before the study commencement. Only patients without mandibular posterior teeth and at least 6 mandibular teeth were to be included in order to standardize RPD framework design.A total of five women (60 to 74 years, mean age 67.6)composed the sample. The study was approved by the institutional ethical review board and the subjects signed informed consent.During a pre-experimental period (approximately 45 days before RPD delivery) supragingival scaling and polishing were performed and oral hygiene instructions (OHI) were given. The OHI included how to properly floss and how to brush the RPD and the teeth with soft toothbrush and specific toothpaste (Colgate Triple Action, Colgate Palmolive Industry Com., Sao Paulo, Brazil). Subjects were instructed to remove the dentures for cleaning after each meal and before sleeping. The shows the fluxogram of the study.The clinical procedures to fabricate maxillary complete dentures and mandibular RPDs were performed by a single professional and followed a strict protocol for the production and placement of the dentures. The cobalt-chromium RPD design was standardized and fabricated by the same dental technician. The clasps used were T-bar (Roach) retainers for direct abutment teeth (canines). The clasps were about 1 mm distant from the gingival edge. The denture bases and/or retainers were connected by a lingual plate that was seated on cingulum rests.At t0 (day 0, baseline), the new maxillary complete denture and mandibular RPD were delivered to patients. Oral hygiene was checked and instructions were repeated at the delivery appointment. Patients were oriented to abstain from any form of chemical control during the study. They also received reinstructions on how to perform tooth brushing as well as denture cleaning.Biofilm was sampled after 1 week (t1) and 4 months (t2) of RPD delivery. All subjects consumed no food or beverages 30 minutes prior to the biofilm collection. First, the RPD was removed from the patient's mouth, washed with saline solution for 10 seconds, and left dry on paper towel for 1 minute. Then, the biofilm adhered on the inner surface of the T-bar clasps was collected with sterile curettes, pooled in a test tube containing 100 µL of DNA extraction solution, and stored at -20℃. rDNA extraction and ethanol precipitation steps followed a previously described protocol. Concentrated rDNA was suspended in 20 µL 10 mM Tris-HCl pH 7.5, 1 mM EDTA solution and PCR-amplified by using universally conserved primers D88 and E94.16 Briefly, 1 µL of each sample was added to 49 µL of reaction mixture containing 5 µL 10 × PCR Buffer (Promega Corporation, Madison, WI, USA), 1.25 unit Taq DNA Polymerase (Promega Corporation), 20 pmol of each primer, and 0.2 mM of each deoxyribonucleotides. PCR amplification was performed in a DNA thermal cycler (PTC-100, MJ Research, Watertown, MA, USA), as reported previously. The PCR products were purified by using spin columns (Wizard Clean-up System, Promega Corporation).A total of 3 µL of the purified PCR products were cloned by using the plasmid vector (TOPO TA Cloning® Kit for Sequencing, Invitrogen Life Technologies, Carlsbad, CA). Transformation used chemically competent cells (One Shot® TOP10 Chemically Competent E. coli, Invitrogen Life Technologies). The next steps for cloning and plasmid DNA purification followed previously described procedures.Purified plasmid DNA was sequenced using primer D88. Sequencing reactions were performed in a DNA thermal cycler (MJ Research PTC-100, MJ Research, Inc., Waltham, MA, USA) using DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences Corp., Piscataway, NJ, USA). Once chain termination was complete, DNA sequencing was carried out in an ABI Prism 3100 Genetic Analyzer (Life Technologies, Carlsbad CA, USA). The sequencing protocol was 95℃, for 30 seconds, followed by 40 cycles of 95℃, 20 seconds; 50℃, 15 seconds, and 60℃, 2 minutes; with a final hold step at 4℃. The resultant 250 sequences presenting 499 or more nucleotides of good quality, which was ascertained by inspecting the chromatograms, were pooled and grouped into two 16S rDNA clone libraries, named t1 and t2. Chimeric sequences were identified using Bellerophon ( and discarded. A total of 180 reminiscent sequences, including 84 from t1 and 96 from t2, were compared with sequences of known phylotypes from GenBank ( using the BLASTn algorithm. Sequence similarity of 98% or greater was used as the definition of a species-level cluster. Clones that were less than 98% similar to the closest known organisms were supposed to represent novel phylotypes. Contiguous sequences were assembled with the Phred/Phrap/Consed software package (, aligned with the CLUSTAL W software. Phylogenetic trees were constructed with MEGA 5 software, according to the calculation of a Jukes and Cantor distance matrix using the neighbor-joining method. Bootstrap confidence values for branching nodes were inferred by generating 1,000 resampling trees. The percentage of coverage was calculated by Good's method with the formula [1 - (n / N)] × 100, where n is the number of phylotypes in a sample represented by one clone (singletons) and N is the total number of sequences in that sample. The computer program ∫- LIBSHUFF ( was used to statistically compare the clone libraries between time points (P ≤ .05). Chao and ACE (Abundance-based Coverage Estimator) statistics were calculated using MOTHUR ( The Chao and ACE estimators are statistical approaches used to estimate the species diversity and richness of microbial communities. Chao calculation considers the number of observed species, the number of singletons (species captured once), and the number of doubletons (species captured twice). The observed species (Sobs) represent the microorganisms collected and identified. The ACE calculation considers the data from all species with fewer than 10 individuals, rather than just singletons and doubletons. Bacteria diversity was considered the dependent variable and time was considered independent variable. The sequences representing novel phylotypes were deposited at GenBank under the following accession numbers KF715649 to KF715662. […]

Pipeline specifications

Software tools BLASTN, Phrap, Consed, Clustal W, mothur
Application Phylogenetics
Organisms Homo sapiens, Fusobacterium nucleatum