Salivary bacterial community profile in normal‐weight and obese adolescent patients prior to orthodontic treatment with fixed appliances

Abstract Objectives The aim of this study was to compare the intra‐oral bacterial profile of normal‐weight and obese adolescents prior to orthodontic treatment with fixed appliances. Materials and methods Nineteen adolescent patients were recruited into two groups based upon body mass index (BMI) and classified as normal‐weight or obese. Unstimulated whole mouth saliva was obtained for 5 minutes. Bacterial DNA extraction was performed from saliva, and 16S rRNA gene sequencing of the V1‐2 variable regions was undertaken followed by analysis using the mothur pipeline. Results Saliva from a total of 19 adolescent patients with mean (SD) age 15.6 (1.8) years were divided into 10 normal‐weight with mean BMI of 19.4 (2.2) kg/m2 and 9 obese with mean BMI of 30.2 (3.5) kg/m2. A total of 156 783 sequences were obtained from the 19 samples with no significant differences in richness or diversity between sample groups by obesity status or gender (AMOVA). The bacterial community in both groups was dominated by bacterial genera characteristic of the human mouth, which included Streptococcus, Porphyromonas, Veillonella, Gemella, Prevotella, Fusobacterium and Rothia. Conclusion There were no differences in alpha or beta diversity of oral bacterial communities between normal‐weight and obese orthodontic patients. Obese adolescents attending for orthodontic treatment had a similar microflora to their normal‐weight counterparts.


| INTRODUC TI ON
Obesity is a global health concern in both child and adult populations. In the United Kingdom alone, the prevalence of obesity has risen ten-fold over the past four decades in both populations. 1 Obesity causes a status of chronic subclinical inflammation in the human body, where adipose tissue can act as an endocrine organ, secreting inflammatory proteins or adipokines that act as both pro-or anti-inflammatory mediators. 2 We have previously demonstrated elevated levels of the pro-inflammatory adipokines leptin and resistin, Whilst increased bacterial richness in the salivary microbiome, more specifically Prevotella and Viellonella, is significantly associated with poorer oral health and reflected amongst patients with increased BMI and old age, 15 other studies have reported no association between BMI and change in salivary microbial array 9,13,16,17 or low bacterial diversity. 18 This is consistent with studies investigating the gut microbiota of obese adult and adolescent individuals, which have found low microbial diversity with lower Bacteroidetes and more Firmicutes compared to normal-weight. 19 There are clear associations between the wearing of orthodontic appliances and oral health, particularly in terms of enamel demineralization, caries and periodontal inflammation. 20 Several studies have reported changes in the salivary microbiome amongst patients receiving orthodontic treatment towards periodontal pathogens, with increases in Gram-positive bacteria from pre-treatment to 1 year 20-22 and in gram-negative anaerobic bacteria, 21,22 which gradually returned from 3 months to 1 year post-treatment. 21 However, prospective data exist to suggest that microbial changes occur towards the end of orthodontic treatment, and if good oral hygiene is maintained, orthodontic treatment does not adversely affect oral health. 23 There is emerging evidence reporting the relationship between the salivary microbiota and obesity, as well as the salivary bacterial community and orthodontics. To date, no studies have examined the salivary microbiome amongst obese adolescents undergoing orthodontic treatment and whether any differences might represent an increased risk for orthodontic iatrogenic effects such as periodontal disease. In this study, the salivary bacterial community in normal-weight and obese subjects were analysed at baseline before the placement of fixed appliances.

| Study design
This cohort study compared the effects of obesity on the intra-oral microbiological flora of adolescent orthodontic patients prior to the placement of fixed appliances. We report and present data according to STROBE (Strengthening the Reporting of Observational Studies in Epidemiology). 24
Sample size was based on data from a previous study investigating the microbial profile in saliva samples from patients diagnosed with periodontitis. 25

| Variables
Baseline data were collected as previously described. 3 Briefly, unstimulated whole mouth saliva (uWMS) was obtained from subjects spitting into a plastic collection tube for 5 minutes and the uWMS rate calculated as millilitre per minute (mL/min). Periodontal health was measured clinically using established validated plaque and gingival indices. 26,27 Subject body weight was measured to the nearest 0.1 kilogram (kg) using a calibrated scale and height measured to the nearest centimetre (cm) using a wall-mounted rule. BMI was calculated as mass (kg) divided by height in metres squared (kg/m 2 ). 3 United Kingdom Royal College of Paediatrics and Child Health World Health Organization growth charts were used to calculate and classify BMI-centile in relation to age and sex. 28 All measurements were taken by a single-trained operator (HS) using the same equipment.

Ethical approval was obtained from the United Kingdom National
Research Ethics Service (UK NRES) (14/LO/0769). Written informed consent was received from all participants.
Bacterial DNA was extracted from saliva samples by means of the GenElute Bacterial Genomic DNA extraction kit (Sigma-Aldrich) following manufacturer's instructions, with an additional lysozyme (45 mg/mL) incubation step for 30 minutes at 37°C. To assess the quantity of nucleic acid, the Qubit fluorometer was utilized to measure DNA (>10 picogram (pg)/mL detection limit) following manufacturer's instructions. In addition, agarose gel electrophoresis (1.5% agarose gel) was performed to assess the integrity of DNA.
For 16S rRNA gene sequencing, a previously published methodology was followed. 29 Polymerase chain reaction (PCR) was performed using fusion primers combining the MiSeq adapter sequences (i5 and i7), an 8-nt barcode sequence, a 10-nt primer pad, a 2-nt linker sequence and a 16S rRNA gene-specific sequence targeting the V1-V2 region of the 16S rRNA genes of bacteria, with near universal specificity. The primer sequences were as follows: Forward PCR primer -

Sequencing was performed by the King's College London Genome
Centre using the Illumina MiSeq instrument and standard protocols for 2x250 basepair (bp) paired-end sequencing.

| Data analysis
Sequences were analysed using the mothur analysis pipeline (version  Table 1.

| PCR and bacterial sequencing data
A total of 156 783 sequences were obtained from the samples after filtering for length and quality, with 4678 sequences sub-sampled from each sample to normalize the data. The richness and diversity of the sample by patient group is shown in Table 2. There were no significant differences in richness or diversity of the samples  (Figures 3 and 4). Obese adolescents attending for orthodontic treatment had a similar microflora to their normal-weight counterparts.

Normalweight Obese
Patients (n) 19 10 9 Male/female (n) 11 Obesity correlates with a number of chronic diseases and inflammatory disorders. 2 The pro-inflammatory activity of adipose tissue can contribute to reduced body immunity and increased susceptibility to infection. 31 The presence of obesity has also been associated with increased risk of dental disease 4,5 with periodontal disease severity positively correlated with increased BMI. 32 Moreover, a recent prospective study revealed significant differences in baseline GCF levels of several pro-inflammatory adipokines and inflammatory mediators in obese subjects compared to normal-weight, suggesting an altered inflammatory state in the periodontal tissues of matched obese adolescents. 3 Orthodontic appliances carry the risk of periodontal tissue damage and enamel decalcification when optimal oral hygiene is not present and maintained. 21  increases this risk has not been investigated. We therefore explored the salivary bacterial community in obese and normal-weight cohorts to investigate potential salivary dysbiosis in obese adolescents that might place them at increased risk of dental disease relevant to orthodontic treatment with fixed appliances.
Current evidence suggesting a difference between the salivary microbiome amongst obese and normal-weight patients is unclear.
It has been reported that obesity can impact on the salivary microbiome in adolescents, with divergence in the bacterial community related to both sex and body mass. 34 17 Similarly, no correlation between BMI and change in salivary bacterial diversity was found in a sample of 25 females, divided into those with polycystic ovary syndrome and healthy groups. 13 Moreover, a more recent study reported a lack of diversity at the species level amongst subjects with Type 2 diabetes, obese and normal-weight adolescents using alpha diversity, although they attributed the lack of correlation to the small sample size in each group. 9 In terms of the influence of orthodontic treatment on the salivary microbiome, a prospective study investigated the effect of fixed and removable orthodontic appliances amongst adolescent patients on salivary bacterial community. They reported significant changes in salivary microbiota composition with increases in S. mutans, lactobacilli and Candida albicans. 37 In addition, the salivary microbiome of 23 subjects undergoing orthodontic treatment showed a significant increase in S. mutans and lactobacilli after insertion of the orthodontic appliances. 38 Therefore, orthodontic appliances create a new environment with shift towards species associated with caries such as S. mutans and lactobacilli as well as periodontitis P. gingivalis, T. forsythia and T. denticola. 22 Whilst alteration in the oral microbiome was reported with significant increase in streptococci, there was no statistical elevation in periodontitisassociated bacteria from pre-treatment to 1 year. 20 A recent randomized clinical study of 120 adolescent subjects who received orthodontic treatment with or without fluoride mouthwash showed that during orthodontic treatment, there was rise in Selenomonas and Porphyromonas counts, whilst Streptococcus, Rothia and Haemophilus were abundant towards the end of orthodontic treatment. They concluded that as long as oral hygiene is maintained, orthodontic treatment does not adversely affect oral health. 23 Our study analyses confirmed lack of statistical difference in the salivary bacterial environment at baseline amongst obese F I G U R E 3 Principal Coordinate Analysis plot comparing bacterial community structure of patient groups by BMI F I G U R E 4 Principal Coordinate Analysis plot comparing salivary bacterial community structure by gender adolescents and their normal lean at the baseline (T1) before insertion