Study design and subjects
Thirty outpatients (16 with ID and 14 without ID) were allocated to the ID and control groups, respectively. All subjects underwent several periodontal examinations and plaque samples were collected in November 2018. The characteristics of the subjects are shown in Table 1. The inclusion criteria for the study included subjects confirmed with “severe ID” based on a nursing certificate issued by the Hiroshima City. The diagnosis was made based on the developmental age of the individuals using multiple development tests and the estimated intelligence quotient was 35–50. The other criteria for inclusion in the study were as follows: age, 20–60 years; presence of permanent dentition; and history of having visited the outpatient clinic at the Department of Special Care Dentistry, Hiroshima University Hospital for regular check-ups after active treatment for dental disease for at least 1 year. The exclusion criteria were being extremely uncooperative while collecting the dental plaque, being under medications (including antibiotics over the past 3 months), and smoking regularly. The hospital treatment records of the individuals for 2016–2018 were reviewed to clarify that the subjects did not have any disease, such as diabetes, autoimmune disease, or infectious disease, that may affect the development of periodontal disease.
Based on a similar study [12], we determined that at least 12 subjects with ID and healthy subjects were needed to ensure an adequate sample size. Initially, 20 patients who seemed to meet the criteria were provided with explanations about the study; subsequently, detailed interviews were taken, and 16 of them were included in the ID group. The control group comprised 14 age- and gender-matched subjects from the outpatient clinics of other departments at the hospital.
Ethical information
This study was approved by the Ethical Committee of the University (Epidemiology-No. E-342), and was registered at the University Hospital Medical Information Network (UMIN) Center (ID 000023503). After having explained the purpose and contents of this study according to ethical guidelines of the Helsinki Declaration (2013), written consent from each participant or guardian was obtained. The subjects were free to withdraw their consent at any time for any reason.
Table 1. Characteristics of the patients
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ID group
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Control group
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P-value
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n
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16
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14
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-
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Gender, n, (%)
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0.4572a
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Male
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11 (68.75)
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7 (50.00)
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Female
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5 (31.25)
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7 (50.00)
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Age, years, mean ± SD
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35.4 ± 8.1
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38.0 ± 12.5
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0.5078b
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Disability, n, (%)
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Intellectual disability
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16 (100.00)
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0 (0.00)
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<0.0001a
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Physical disability
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1 (6.25)
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0 (0.00)
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<0.0001a
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Number of teeth, mean ± SD
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26.4 ± 2.3
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28.0 ± 0.00
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0.0060c
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DMFT, mean ± SD
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11.1 ± 8.0
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8.5 ± 5.5
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0.3025b
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PMA index, median, [IQR]
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22.0 [20.5, 26.0]
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2.5 [1.0, 4.0]
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<0.0001b
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PI, median, [IQR]
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1.9 [1.0, 2.2]
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0.4 [0.0, 0.2]
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<0.0001b
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GI, median, [IQR]
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1.6 [1.3, 1.7]
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0.1 [0.0, 0.4]
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<0.0001b
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PD, mean ± SD
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2.8 ± 0.3
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2.1 ± 0.2
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<0.0001b
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a Fisher's exact test, b unpaired t-test, and c Mann–Whitney U test.
Values are mean ± SD unless otherwise noted.
DMFT, Number of decayed, missing, and filled teeth; IQR, Interquartile range; PI, plaque index); GI, gingival index; PD, probing depth; SD, standard deviation; ID, intellectual disability; PMA, papillary-marginal-attached
Clinical periodontal parameters measurements
Oral and periodontal examinations were performed by a well-experienced dentist under artificial lighting with the patient lying supine on the dental chair. The DMFT score, PMA index [18], PI [19], GI [20], and PD were recorded. The DMFT score was calculated as the total number of carious (D), extracted (M), and restored (F) teeth per person. The PMA index was evaluated by visual inspection of the anterior gingival units (papillary, marginal, and attached gingiva) around the erupted teeth based on the degree of redness and swelling, without the aid of an instrument. The PMA index was graded as follows: grade 0, no inflammation; and grade 1, presence of inflammation. The GI was graded as follows: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation. The GI and PD were assessed using a calibrated periodontal probe (UNC-15; Hu-Friedy, Chicago, IL, USA) on four aspects (mesiobuccal, distobuccal, mesiolingual, and distolingual) of six teeth (maxillary right first molar, maxillary right lateral incisor, maxillary left first premolar, mandibular right first premolar, mandibular left lateral incisor, and mandibular left first molar).
Intra-examiner reliability
To assess the reproducibility of the PD, 10 patients with two contralateral teeth and a periodontal pocket depth of 6 mm or more who did not participate in the study were used. The patients were evaluated by the investigators three times at three-day intervals with sufficiently reproducible PD measurements (grand mean, 2.2; change in mean, 0.07; typical error, 0.30; repeatability coefficient, 0.84; and intraclass correlation coefficient, 0.63). The reproducibility of the PMA index, PI, and GI were assessed as described for PD, showing a sufficiently high repeatability (data not shown).
Collection of oral samples and DNA extraction
Dental plaque was collected by thoroughly brushing all the teeth for 1 min using a sterile toothbrush [13,14] followed by the rinsing of the toothbrush in 15 mL of distilled water for 30 s. The obtained samples were placed in tubes and immediately sent to the J-Bio 21 Center laboratory (Nippon Steel & Sumikin Eco-tech Corp., Japan), where the genomic DNA was extracted and stored at −20°C. DNA extraction, polymerase chain reaction (PCR) analysis, next-generation sequencing, and analysis using the Quantitative Insights Into Microbial Ecology (QIIME) [21] software package were all performed in the same laboratory. Genomic DNA was extracted from a 0.5-g sediment sample using the Extrap Soil DNA Kit Plus, ver.2 (J-Bio21 Center, Tsukuba, Japan).
PCR amplification and DNA sequencing of bacterial 16S rRNA
The extracted DNA was quantified using a PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, USA), and the number of 16S rRNA gene copies in the extracted DNA was determined by quantitative PCR assay using a Bac1115 Probe quenching probe and primers Bac 1055YF and Bac1392R [22]. The Bac1115Probe was labeled with boron-dipyrromethene at the 3’-end and a quenching fluorescence dye (Qprobe; J-Bio21 Center, Tsukuba, Japan) at the 5’-end. All the adenine nucleotides of the Bac1115Probe were modified with locked nucleic acids. Quantitative PCR assays were performed using a Rotor-Gene Q (Qiagen, Valencia, CA, USA) with the following parameters: initial denaturation at 93°C for 2 min, followed by 50 cycles of 15 s at 93°C and 20 s at 61°C. Thereafter, the number of 16S rRNA gene copies per gram in each sample was calculated for each sediment sample. The U515F (5’-GTGYCAGCMGCCGCGGTA-3’) and 926R (5’-CCGYCAATTCMTTTRAGTT-3’) primer sets were used to amplify the V4–V5 region of the bacterial 16S RNA gene [21]. PCR product sequencing was performed with an Illumina MiSeq instrument (Illumina KK, Tokyo, Japan) and sequence data were analyzed using QIIME [23]. Approximately 30,000 430-bp reads were obtained for each sample after filtration (minimum quality value, 25) and removal of the chimeras. Sequences with >97% similarities were then clustered in operational taxonomic units (OTUs) using UCLUST with the furthest algorithm [24]. The most abundant reads from each cluster were assigned at the representative sequences. The OTU representatives were subjected to phylogenetic analysis using the Greengenes [25] and Living tree of Silva [26] databases.
The relative abundance of bacterial 16S rRNA gene copies for each OTU in the sediment samples (copies g-sediment−1) were evaluated by multiplying the total number of 16S rRNA gene copies per gram of sediment by the number of target bacterial sequence read OTUs and dividing by the total number of sequence reads.
OTUs related to bacteria capable of utilizing azo dyes and aniline (as a representative aromatic amine) were analyzed against the KEGG [27] and UniProt (UniProt Consortium, 2011) databases. In order to facilitate quantitative comparisons with data for gene copy number among the samples collected over 3 years, OTU sequences related to bacteria capable of utilizing azo dye and aniline were represented as 16S rRNA gene copies per g of sediment.
Statistical analysis
The sample size was derived by power estimation as described in a previous study, which evaluated the difference in the percentage of periodontal pathogens in the subgingival biofilm between healthy individuals and patients with chronic periodontitis [28]. The values for continuous variables with non-normal distribution are expressed as median and interquartile range (IQR), while those with normal distribution are expressed as mean and standard deviation (SD); the values for categorical variables are presented as number and percentage. An unpaired t-test was used to compare continuous variables with normal distribution, while Mann–Whitney U test was used for variables with non-normal distribution and the Fisher’s exact test for nominal variables. Endpoint analysis was based on an analysis of covariance model, with the test group as effect and the baseline value as a covariate. We calculated the least square mean (LSM) of the difference in the relative abundance of each bacteria between two groups using the baseline abundance rate as a covariate. The SAS 9.4 software package (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses, and p-values <0.05 indicated statistical significance.