Towards Microbiome Transplant as a Therapy for Periodontitis
Adult subjects were recruited at the Department of Periodontics, University of Washington, Seattle, WA, U.S.A. and Section of Periodontics, University of Düsseldorf, Germany. A written informed consent for participation in the study was obtained from the subjects. The study was approved by the University of Washington Institutional Review Board, ref. number 3570. Subjects were enrolled if they had one of the following clinical conditions: severe periodontitis, caries, edentulism, or oral health. A periodontitis case was defined as having at least 2 interproximal sites at different teeth with clinical attachment loss (CAL) of 6 mm or greater and at least 1 interproximal site with probing depth (PD) of 5 mm or greater and a minimum of 20 permanent teeth, not including 3 molars. Subjects were excluded from the periodontitis group if they had multiple established caries lesion or wore a removable partial denture. A caries case was defined as having the following number of teeth with established caries lesions: 6 or more teeth in subjects 20 to 34 years of age; 4 or more teeth in subjects 35 to 49 years of age; and 3 or more teeth in subjects 50 years of age and older. Established caries was defined as a class 4 lesion according to the International Caries Detection and Assessment System. The number of teeth with caries lesion in caries cases was greater than one standard deviation above the mean of caries extent in respective age group the U.S.A.. Exclusion criteria for a caries case were interproximal sites with CAL of 4 mm or greater or PD of 5 mm or greater. An edentulous case had to be completely edentulous in both jaws and their teeth had to be extracted more than one year before the enrollment in the study. A healthy case was defined as having 28 teeth, not counting 3 molars, or 24 or more teeth, not counting 3 molars if premolars had been extracted for orthodontic reasons or were congenitally missing with no signs of oral disease. Exclusion criteria for a healthy case included: smoking, loss of permanent teeth due to caries or periodontitis, any interproximal sites with CAL of 4 or greater or PD of 5 mm or greater, or any established caries lesions. Exclusioncriteria for all groups included: oral mucosal lesions, systemic diseases, and use of antibiotics or local antiseptics within 3 months prior to the study.
For all but the edentulous patients, supra- and subgingival plaque was collected from six sites with the deepest probing depth in each sextant. One sterile paper point per site was inserted into the deepest aspect of the periodontal pocket or gingival sulcus. Biofilm from oral mucosae was collected by swiping a sterile cotton swab over the epithelial surfaces of the lip, left and right buccal mucosae, palate, and dorsum of the tongue. Samples were stored at −80 °C.
Microbial DNA was isolated from cells by physical and chemical disruption using zirconia/silica beads and phenol-chloroform extraction in a FastPrep-24 bead beater. Prokaryotic 16S rRNA genes were amplified using universal primers (27 F and 1392R) using the GemTaq kit from MGQuest (Cat# EP012). The PCR program involved a pre-amplification step of 10 cycles with annealing temperature of 56 °C followed by 20 amplification cycles with annealing temperature 58 °C. In each cycle, elongation time was 1 min 10s, at 72 °C. PCR was finalized by extended elongation for 5 min. PCR products were purified with DNA Clean & Concentrator columns (Zymo Research, USA) and quantified using the NanoDrop (Agilent, USA).
Due to technical reasons, for some subjects, subgingival and mucosal microbiotas were pooled together into one vial, while for other subjects the subgingival and mucosal microbiotas were stored separately. Since our goal was to investigate the entire microbial community, for the separately stored supra and subgingival microbiota samples, equal quantities of PCR product derived from swab and paper point samples were pooled together for each patient. For edentulous patients, there were no paper point samples. Each purified PCR product, 500 ng, was labeled with a Multiplex Identifier (MID) during the Roche Rapid Library preparation step. Four MID-tagged sequences, representing each of the conditions, were combined in equimolar concentrations and subjected to emPCR and DNA sequencing protocols as specified by the manufacturer's recommendations for the Roche 454 Jr. instrument.
The obtained sequences were separated out by their respective Multiplex Identifier (MID) and uploaded to the MG-RAST web server. The MG-RAST pipeline assessed the quality of sequences, removed short sequences (multiplication of standard deviation of length cutoff of 2.0) and removed sequences with ambiguous bp (non-ACGT; maximum allowed number of ambiguous base pair was set to 5). The pipeline annotated the sequences and allowed the integration of the data with previous metagenomic and genomic samples. The RDP database was used as annotation source, with minimum sequence identity of 97 %, maximum e-value cutoff at 10 , and minimum sequence length of 100 bases. Alpha diversity analysis was conducted in MG-RAST.
Orthogonal transformation of the annotated rRNA genes to their principal components (PC) was conducted using normalized abundances. Normalization of the abundance was performed identically to the procedure used by MG-RAST. Specifically, abundances were increased by one, log2 transformed, and centered to produce relative values. Relative values were standardized by dividing them by the standard deviation of the log2 values. The data were graphed on a 2 dimensional ordination plot. To determine the relative contribution of the microbial species to the plot, we let X denote the 16 by 578 (patients by species) matrix of the normalized abundance values. The matrix X was used to produce a 16 × 16 matrix D of distances between all pairs of subjects. Principal coordinate analysis (PCoA, i.e., multidimensional scaling) was achieved by performing principal component analysis (PCA) on the matrix of distances, D. The Euclidean distance metric was used to create this matrix, but this metric produced results similar to those when the alternative Bray-Curtis distance was used. To investigate and visualize differences between the four patient groups, the first two principal components, PC1 and PC2, of the distance matrix D were retained. To establish which species were most prominently responsible for the groupings, the projection of each species onto the (PC1, PC2) plane was calculated; those species with the largest projections are displayed in the right panel of Fig. 3. That is, this figure shows a biplot of the species most highly correlated PC1 and PC2.
Mann–Whitney test (alpha = 0.05) was used to investigate significant difference in normalized relative abundances. The abundance data were normalized in two different ways: MG-RAST normalization as described above and raw abundances normalized to the total number of reads in a sample. Differences in alpha diversities by condition were determined using ANOVA. Two-tailed T-tests, assuming unequal variance, were used investigate if there was significantly different in the means (alpha = 0.05). These analyses were performed using SAS JMP.
According to previously suggested concentration, sodium hypochlorite experiments were conducted with a 1:50 dilution of the 6 % household bleach (Chlorox, The Clorox Company, USA), the "hypochlorite working solution". The dilution corresponds to 16 mM NaOCl. The neutralizing agent was a sodium ascorbate buffer produced by adjusting pH of a 23 mM ascorbic acid solution with concentrated NaOH until pH 5.3.
Three samples of dental plaque from a volunteer was subjected to three challenges: (i) resuspension in saline, (ii) resuspension in the hypochlorite working solution and (iii) resuspension in the hypochlorite working solution that was previously neutralized with equal volume of the ascorbate buffer. Resuspended plaque samples were plated on blood agar plates.
Active chlorine concentration was crudely assessed by iodine colorimetry. A colorimetric solution contained 1 volume of 0.1 % stabilized starch solution (Fisher Scientific) mixed with 0.1 volume of 1 M potassium iodide. Two volumes of the colorimetric solution were mixed with 1 volume of the solution containing NaOCl. Intense blue color indicated detectable active chlorine. Iodine colorimetry was calibrated with a dilution series of the hypochlorite working solution with the following dilutions 1:200, 1:400, 1:800, 1:1600, 0. The dilution1:800 still showed a hint of blue, while 1:1600 was completely colorless.
Methods
Study Subjects
Adult subjects were recruited at the Department of Periodontics, University of Washington, Seattle, WA, U.S.A. and Section of Periodontics, University of Düsseldorf, Germany. A written informed consent for participation in the study was obtained from the subjects. The study was approved by the University of Washington Institutional Review Board, ref. number 3570. Subjects were enrolled if they had one of the following clinical conditions: severe periodontitis, caries, edentulism, or oral health. A periodontitis case was defined as having at least 2 interproximal sites at different teeth with clinical attachment loss (CAL) of 6 mm or greater and at least 1 interproximal site with probing depth (PD) of 5 mm or greater and a minimum of 20 permanent teeth, not including 3 molars. Subjects were excluded from the periodontitis group if they had multiple established caries lesion or wore a removable partial denture. A caries case was defined as having the following number of teeth with established caries lesions: 6 or more teeth in subjects 20 to 34 years of age; 4 or more teeth in subjects 35 to 49 years of age; and 3 or more teeth in subjects 50 years of age and older. Established caries was defined as a class 4 lesion according to the International Caries Detection and Assessment System. The number of teeth with caries lesion in caries cases was greater than one standard deviation above the mean of caries extent in respective age group the U.S.A.. Exclusion criteria for a caries case were interproximal sites with CAL of 4 mm or greater or PD of 5 mm or greater. An edentulous case had to be completely edentulous in both jaws and their teeth had to be extracted more than one year before the enrollment in the study. A healthy case was defined as having 28 teeth, not counting 3 molars, or 24 or more teeth, not counting 3 molars if premolars had been extracted for orthodontic reasons or were congenitally missing with no signs of oral disease. Exclusion criteria for a healthy case included: smoking, loss of permanent teeth due to caries or periodontitis, any interproximal sites with CAL of 4 or greater or PD of 5 mm or greater, or any established caries lesions. Exclusioncriteria for all groups included: oral mucosal lesions, systemic diseases, and use of antibiotics or local antiseptics within 3 months prior to the study.
Sample Collection
For all but the edentulous patients, supra- and subgingival plaque was collected from six sites with the deepest probing depth in each sextant. One sterile paper point per site was inserted into the deepest aspect of the periodontal pocket or gingival sulcus. Biofilm from oral mucosae was collected by swiping a sterile cotton swab over the epithelial surfaces of the lip, left and right buccal mucosae, palate, and dorsum of the tongue. Samples were stored at −80 °C.
Molecular Methods
Microbial DNA was isolated from cells by physical and chemical disruption using zirconia/silica beads and phenol-chloroform extraction in a FastPrep-24 bead beater. Prokaryotic 16S rRNA genes were amplified using universal primers (27 F and 1392R) using the GemTaq kit from MGQuest (Cat# EP012). The PCR program involved a pre-amplification step of 10 cycles with annealing temperature of 56 °C followed by 20 amplification cycles with annealing temperature 58 °C. In each cycle, elongation time was 1 min 10s, at 72 °C. PCR was finalized by extended elongation for 5 min. PCR products were purified with DNA Clean & Concentrator columns (Zymo Research, USA) and quantified using the NanoDrop (Agilent, USA).
Due to technical reasons, for some subjects, subgingival and mucosal microbiotas were pooled together into one vial, while for other subjects the subgingival and mucosal microbiotas were stored separately. Since our goal was to investigate the entire microbial community, for the separately stored supra and subgingival microbiota samples, equal quantities of PCR product derived from swab and paper point samples were pooled together for each patient. For edentulous patients, there were no paper point samples. Each purified PCR product, 500 ng, was labeled with a Multiplex Identifier (MID) during the Roche Rapid Library preparation step. Four MID-tagged sequences, representing each of the conditions, were combined in equimolar concentrations and subjected to emPCR and DNA sequencing protocols as specified by the manufacturer's recommendations for the Roche 454 Jr. instrument.
Data Analysis
The obtained sequences were separated out by their respective Multiplex Identifier (MID) and uploaded to the MG-RAST web server. The MG-RAST pipeline assessed the quality of sequences, removed short sequences (multiplication of standard deviation of length cutoff of 2.0) and removed sequences with ambiguous bp (non-ACGT; maximum allowed number of ambiguous base pair was set to 5). The pipeline annotated the sequences and allowed the integration of the data with previous metagenomic and genomic samples. The RDP database was used as annotation source, with minimum sequence identity of 97 %, maximum e-value cutoff at 10 , and minimum sequence length of 100 bases. Alpha diversity analysis was conducted in MG-RAST.
Orthogonal transformation of the annotated rRNA genes to their principal components (PC) was conducted using normalized abundances. Normalization of the abundance was performed identically to the procedure used by MG-RAST. Specifically, abundances were increased by one, log2 transformed, and centered to produce relative values. Relative values were standardized by dividing them by the standard deviation of the log2 values. The data were graphed on a 2 dimensional ordination plot. To determine the relative contribution of the microbial species to the plot, we let X denote the 16 by 578 (patients by species) matrix of the normalized abundance values. The matrix X was used to produce a 16 × 16 matrix D of distances between all pairs of subjects. Principal coordinate analysis (PCoA, i.e., multidimensional scaling) was achieved by performing principal component analysis (PCA) on the matrix of distances, D. The Euclidean distance metric was used to create this matrix, but this metric produced results similar to those when the alternative Bray-Curtis distance was used. To investigate and visualize differences between the four patient groups, the first two principal components, PC1 and PC2, of the distance matrix D were retained. To establish which species were most prominently responsible for the groupings, the projection of each species onto the (PC1, PC2) plane was calculated; those species with the largest projections are displayed in the right panel of Fig. 3. That is, this figure shows a biplot of the species most highly correlated PC1 and PC2.
Mann–Whitney test (alpha = 0.05) was used to investigate significant difference in normalized relative abundances. The abundance data were normalized in two different ways: MG-RAST normalization as described above and raw abundances normalized to the total number of reads in a sample. Differences in alpha diversities by condition were determined using ANOVA. Two-tailed T-tests, assuming unequal variance, were used investigate if there was significantly different in the means (alpha = 0.05). These analyses were performed using SAS JMP.
Sodium Hypochlorite Experiments
According to previously suggested concentration, sodium hypochlorite experiments were conducted with a 1:50 dilution of the 6 % household bleach (Chlorox, The Clorox Company, USA), the "hypochlorite working solution". The dilution corresponds to 16 mM NaOCl. The neutralizing agent was a sodium ascorbate buffer produced by adjusting pH of a 23 mM ascorbic acid solution with concentrated NaOH until pH 5.3.
Three samples of dental plaque from a volunteer was subjected to three challenges: (i) resuspension in saline, (ii) resuspension in the hypochlorite working solution and (iii) resuspension in the hypochlorite working solution that was previously neutralized with equal volume of the ascorbate buffer. Resuspended plaque samples were plated on blood agar plates.
Active chlorine concentration was crudely assessed by iodine colorimetry. A colorimetric solution contained 1 volume of 0.1 % stabilized starch solution (Fisher Scientific) mixed with 0.1 volume of 1 M potassium iodide. Two volumes of the colorimetric solution were mixed with 1 volume of the solution containing NaOCl. Intense blue color indicated detectable active chlorine. Iodine colorimetry was calibrated with a dilution series of the hypochlorite working solution with the following dilutions 1:200, 1:400, 1:800, 1:1600, 0. The dilution1:800 still showed a hint of blue, while 1:1600 was completely colorless.