Search strategy and screening process
The search details are provided in the PRISMA flow chart (Fig. 1). The search strategy resulted in 2077 potential articles: 725 articles from PubMed (MEDLINE), 872 articles from Scopus, 480 articles from the Dentistry and Oral Sciences database, and one article from reference searching [26]. After removal of duplicates, 1627 articles were screened. All articles were assessed according to the inclusion and exclusion criteria and this led to full text analysis of 22 articles [10, 11, 26–45]. One study did not report the complete sequencing of the subgingival microbiome [36] and one study did not report baseline data [45] and while one study assessed on healthy participants [30] and were all 3 thus excluded. Overall, 19 studies were included for descriptive analysis and 10 studies [10, 11, 27–29, 32, 34, 35, 37, 44] were meta-analysed.
Characteristics of included studies
Characteristics of the included studies were examined in the context of study design, periodontal disease definition, age, description of test and control group, plaque collection method, hypervariable region of 16S rRNA gene and follow up period (Table 1).
Table 1
Characteristic of Included Studies
First author and Year
|
Study design
|
Disease
|
Disease definition
|
Age
|
Case
(n)
|
Control (n)
|
Test description
|
Control description
|
Site and plaque collection method
|
Hypervariable region of 16S rRNA gene
|
Follow up
|
Final outcome
|
Kruse et al. 2020
|
Single-blinded, randomised controlled split mouth study
|
Chronic periodontitis patient undergoing maintenance therapy
|
2 single-rooted with PPD = 5 mm and positive BOP or > 5 mm with or without positive BOP
|
61.4 ±10.6
|
10
|
Air polishing using trehalose powder
|
Ultrasonic scaling
|
Subgingival plaque
Paper point
|
NA
|
3 months
|
Air polishing and sonic treatment have a similar effect on the subgingival microbiome.
|
Al-Kamel et al. 2019
(Prevention study Yemen)
|
Triple blinded,
placebo-controlled, parallel-arm RCT
|
Experimental gingivitis
|
21 days gingivitis model
|
22.22 ± 1.73
|
10
|
10
|
T1:1.25% NAC mouthwash
T2: 0.2% CHX mouthwash
|
Placebo mouthwash
|
Subgingival plaque
Paper point
|
V1-V3 region
|
3 weeks
|
CHX was more effective in preventing and reversing experimental gingivitis than NAC. There was a non-significant increase in the species richness and alpha diversity compared to baseline for placebo and NAC group while CHX resulted in decrease in these parameters.
|
Al-Kamel et al. 2019 (Treatment study) Yemen
|
10
|
10
|
1.25% N-acetyl cysteine mouthwash
|
0.2% CHX mouthwash
|
Subgingival plaque
Paper point
|
V1-V3 region
|
3 weeks
|
CHX use was associated with significant decrease in species richness and alpha diversity while NAC caused no change.
|
Hagenfeld et al. 2019 Germany
|
Double-blind, two centre RCT
|
Mild to moderate periodontitis
|
PPD of ≥ 4 mm in at least four teeth except third molars
|
54.22
|
20
|
21
|
Toothpaste containing zinc substituted carbonated hydroxyapatite
|
Toothpaste with amine fluoride/stannous fluoride
|
Subgingival plaque
Paper point
|
V4-V6 region
|
12 weeks
|
The toothpaste containing anti-adhesive HA did not induce any changes on the microbial composition compared to anti-adhesive and antimicrobial AmF/SnF2. There was no changes in the alpha diversity.
|
Lu et al. 2019 China
|
Clinical trial
|
Periodontitis patients on maintenance therapy
|
Maintained subjects that had stable condition with more than 20 teeth, bop (%) ≤ 25%, PPD ≤ 5 mm
|
50.18 ± 12.08
|
17
|
17
|
Ultrasonic scaling
|
Air polishing
|
Subgingival plaque
Sterile Gracey curettes
|
V3- V4 region
|
12 weeks
|
There was reduction in the microbial diversity, proportion of periodontitis associated bacteria and pathogenic metabolism after the use of ultrasonic (US) and air polishing (AP). Chao1 was significantly reduced at 2 weeks in both US and AP group but increased significantly from week 2 to 8 to week 12. Similarly Shannon index decreased after treatment and then increased at week 12.
|
Woelber et al. 2019 Germany
|
Single blinded RCT
|
Gingivitis
|
Mean GI index ≥ 0.5
|
27.2 (4.7)
|
15
|
15
|
Anti-inflammatory diet low in processed carbohydrates and animal proteins and rich in omega-3 fatty acids, vitamin c, vitamin d, antioxidants, plant nitrates and fibre
|
Western diet
|
Subgingival plaque
Sterile Gracey curettes
|
V3-V4 region.
|
6 weeks
|
There was no significant difference seen in the subgingival microbiome with both the diets. The alpha diversity was slightly higher in experimental group.
|
Hagenfeld et al. 2018 Germany
|
Double-blind, parallel group RCT
|
AAP 1999 definition for moderate/severe chronic periodontitis
|
3mm to < 5mm/ _5mm attachment loss
|
NA
|
42
|
47
|
SRP + 50 mg amoxicillin and 400 mg metronidazole
|
SRP + placebo
|
Subgingival plaque
Paper points
|
V3-V4 region
|
8 weeks
|
Adjunctive use of antibiotics with SRP significantly reduced the richness, decreased the periodontitis associated genera and changed the bacterial compositional structure.
|
Chen et al. 2018 USA
|
Pre/post intervention
|
Chronic periodontitis
|
NA
|
NA
|
19
|
SRP
|
Before SRP
|
Subgingival plaque
Paper point
|
V4 region
|
4 weeks
|
SRP was effective in decreasing the relative abundances of periodontitis associated bacteria.
|
Belstrom et al. 2018 Denmark
|
Pre/post intervention
|
AAP 2015 definition for moderate/severe chronic periodontitis
|
PD ≥ 5 & <7 mm and CAL 3–5 mm/ PD ≥ 7 mm and CAL ≥ 5 mm
|
47–75
|
25
|
SRP
|
Before SRP
|
Subgingival plaque
Sterile Gracey curette
|
V3-V4 region
|
12 weeks
|
After SRP there was a decrease in the relative abundance of periodontitis associated genera in the subgingival plaque. There was a decrease in alpha diversity significantly after 2 and 6 weeks.
|
Queiroz et al. 2017 Brazil
|
Single blinded RCT
|
Chronic periodontitis with Class II furcation on molars
|
Horizontal furcation PD ≥ 4 mm
|
53.14
|
13
|
13
|
Beta-tricalciumphosphate/hydroxyapatite graft/ EMD + BONE
|
EMD only
|
Subgingival plaque
Paper point
|
NA
|
6 months
|
Treatment with EMD alters the dysbiotic subgingival microbiome and decreases the pathogen richness and increases commensal abundance.
|
Han et al. 2017 China
|
Pre/post intervention
|
AAP 1999 definition for generalized Aggressive Periodontitis
|
Rapid attachment loss and bone destruction, and possible familial aggregation of disease.
|
28 ± 1.41
|
2
|
SRP
|
No treatment
|
Subgingival plaque
Sterile Gracey curettes
|
V4 region
|
1 month
|
There was lower Shannon index and higher Chao1 index post treatment. Bacteroidetes, Spirochetes and Fusobacteria were related to pathogenicity while Actinobacteria and Proteobacteria were associated with resolution of clinical symptoms.
|
Liu et al. 2017 China
|
Pre/post intervention
|
AAP 1999 definition for generalized Aggressive Periodontitis
|
Rapid attachment loss and bone destruction, and possible familial aggregation of disease.
|
30.75 ± 3.17
|
12
|
SRP
|
No treatment
|
Subgingival plaque
Filter paper
|
V3-V4 region
|
6 weeks
|
SRP was effective in reducing alpha diversity, changing bacterial compositional structure, reducing relative abundance and prevalence of certain bacterial species.
|
Califf et al. 2017
USA
|
Clinical trial
|
Periodontitis
|
At least four separate teeth with a pocket depth of
6 mm
|
41
|
17
|
17
|
0.25% sodium hypochlorite
|
Water rinse
|
Subgingival plaque
Sterile Gracey curette
|
V4-V5
|
3 months
|
High diversity and number of metabolites were both significantly related to periodontal deep pockets. For effectiveness of treatment metabolic dynamisms was more indicative than shifts in composition in community.
|
Bizzaro et al. 2016 Netherland
|
Single blinded RCT
|
Chronic periodontitis
|
Proximal attachment loss of ≥ 3 mm at ≥ 2 non-adjacent teeth + below median responders
|
NA
|
9
|
10
|
SRP + 0.12% CHX rinse (2 × day × 28 days) and antibiotics (amoxicillin 375 mg + metronidazole 250 mg, 3 times a day × 7 days)
|
SRP + CHX 0.12% only
|
Subgingival plaque
Paper point
|
V5-V7 region
|
12 months
|
Antibiotic usage after 3 months resulted in significant changes in the subgingival microbiome. SRP + CHX predicted better clinical outcome.
|
Huang et al. 2016
China
|
Double-blind randomised controlled clinical trial
|
Moderate gingivitis
|
At least 10 bleeding sites; mean
MGI is from 1.0–2.5
|
33.80 ± 7.86
|
47
|
44
|
The brush-plus-rinse group:
manual toothbrush and a toothpaste containing
0.321% sodium fluoride and 1.16% stannous chloride and 20 ml rinse with 0.0747% CPC for 30 seconds after brushing.
|
Brush twice-daily with a manual brush and a 0.243% sodium fluoride toothpaste
|
Supragingival plaque
Sterile Gracey curette
|
V1-V3 region
|
27 days
|
Brush-plus-rinse group exhibited more profound temporal changes in both α and β diversity of plaque microbiota
than brush-alone group.
|
Teng et al. 216
|
Double blind randomised controlled trial
|
Experimental gingivitis
|
21 days gingivitis model
|
18–53
|
41
|
50
|
CPC mouthrinse, two times daily
|
Water rinse
|
Supragingival Sterile Gracey curette
|
V1-V3 region
|
3 weeks
|
The α and β diversity revealed that CPC treatment prevents acquisition of new taxa that would
otherwise accumulate but maintains the original biodiversity of healthy plaques.
|
Shi et al. 2015
|
Pre/post intervention
|
Chronic periodontitis
|
N/A
|
53
|
17
|
SRP
|
Before SRP
|
Subgingival plaque
Sterile Gracey curettes
|
Shotgun
|
4–19 weeks
|
After SRP there was a decrease in alpha diversity, change in composition towards health associated bacteria, lowered diseased associated functional pathway and reduction in microbial correlation.
|
Laksmana et al. 2012
USA
|
Pre/post intervention
|
AAP definition of Aggressive Periodontitis
|
Rapid attachment loss and bone destruction, and possible familial aggregation of disease.
|
N/A
|
2
|
SRP + 500mg amoxicillin + 500mg metronidazole, three times daily x 8 days
|
N/A
|
Subgingival plaque
Paper point
|
V4 region
|
8 weeks
|
The subgingival microbiome changed after therapy. After intervention there was an increase in the gram positive and gram negative commensals while a decrease in the bacteria of the red complex.
|
Junemann et al. 2012
Germany
|
Double blind, parallel group, placebo controlled RCT
|
Generalized severe chronic periodontitis
|
More than 38% of sites with pocket probing depths of 6 mm or more
|
N/A
|
2
|
2
|
SRP + 500mg amoxicillin + 400mg metronidazole, three times daily x 7 days
|
SRP
|
Subgingival plaque
Paper point
|
V6 region
|
2 months
|
For the both the intervention group, there was increase in alpha diversity. There was shift in bacterial composition from Gram-negative Bacteroidetes to Gram- positive bacteria.
|
Yamanaka et al. 2012
Japan
|
Pre/post intervention
|
Periodontitis
|
N/A
|
35–73
|
19
|
SRP
|
Before SRP
|
Supragingival Sterile curettes
|
V1-V2 region
|
Immediately
|
Periodontal therapy decreased microbial richness and biodiversity in the plaque microbiota
|
RCT: Randomized clinical trial; SRP: Scaling and root planing; PPD: periodontal probing depth; CAL: clinical attachment level; BOP: Bleeding on probing; NAC: N-acetyl cysteine CHX: Chlorhexidine; GI: Gingival index; MGI: Mazza Gingival index; AAP: American Association of Periodontology; CPC: Cetylpyridinium Chloride; EMD: emodogain |
Study type and intervention:
Of the 19 studies, only half were RCT [10, 27–29, 31, 32, 34, 37, 40, 44], whereas seven studies [11, 26, 33, 38, 39, 42, 43] were pre and post intervention studies (before and after SRP) and two studies [35, 41] were clinical trials without clearly defining randomisation. For the purposes of this analysis, one RCT [10] was split into three sub-studies; two prevention and one treatment sub-study, while another clinical trial [27] described their results as above or below median responses; thus we analysed this data separately. In total, six types of interventions were observed: (a) six studies [11, 26, 33, 38, 42, 43] were single arm studies comparing pre and post SRP; (b) four studies [27, 29, 39, 40] supplemented SRP with antibiotics; (c) four studies [10, 28, 37, 41] used mouthwash rinses only; (d) two studies [35, 44] compared ultrasonic scaling to air polishing; (e) one study [32] used toothpastes with active ingredients; (f) one study used a regenerative material (Beta-tricalcium phosphate/EMD/Hydroxyapatite) for furcation therapy; and (g) one study used an anti-inflammatory diet. The duration of the interventions ranged from immediately after treatment [43] to three weeks, [10, 28], 4 weeks [26, 37, 38], 6 weeks [11, 34], 8 weeks [29, 39, 40], 12 weeks [32, 33, 35, 41, 44], 6 months [31] to 12 months [27].
Study participants:
All the study subjects were generally systematically healthy patients and the age ranged from 18–73 years. All the studies included both the male and female gender except for one study [38] that only included females. For this review, we included studies on all forms of periodontal diseases, including generalised gingivitis [34, 37], chronic periodontitis [26, 27, 29, 31–33, 40, 42, 44], aggressive periodontitis [11, 38, 39], periodontitis (unclassified) [35, 41, 43] and experimental gingivitis [10, 28].
Study methodology and metagenomics analysis:
The plaque collection methods varied across studies, the most popular methods for collection of plaque was sterile paper points [10, 26, 27, 29, 31, 32, 39, 40, 44] and sterile gracey curettes [28, 33–35, 37, 38, 41–43]. Only one author used filter paper for plaque collection [11]. All the included studies used amplicon sequencing of 16S rRNA except for one study that used shotgun approach for sequencing. The most commonly used hypervariable region observed was the V3-V4/ V4 [11, 26, 29, 33–35, 38, 39] region of 16S rRNA, while some authors used V1-V3 [10, 28, 37, 43], V4-V6 [32], V4-V5 [41] V5-V7 [27] and V6 [40]. All the studies used operational taxonomic units as the basis of metagenomics analysis, except for two studies [29, 32] that used ribosomal sequencing variants (RSVs).
Measures of Alpha diversity:
Alpha diversity is a measure of within sample diversity of community, described in terms of the number (richness) or distribution (evenness) [46]. The bacterial richness (number of observed species) was assessed by three authors [10, 11, 28, 29, 32, 40, 43, 44]. The commonly used indices to measure alpha diversity was Shannon Diversity Index [10, 11, 26–29, 37, 38, 40, 42, 43]. Some studies used Chao1 index [10, 11, 35, 38, 41, 43], Simpson index[11, 26, 40], Pilous evenness [29, 40] and Faith’s phylogenetic diversity [41].
Measure of Beta diversity:
Beta diversity is a measure of the between sample differences of pairs of communities. The beta diversity was observed by all studies except for four studies [31, 33, 39, 40]. It can be measured either using principal coordinates analysis (PCoA) or principal component analysis (PCA) [46]. To assess the beta-diversity, PCoA was used by 11 studies [10, 11, 29, 32, 35, 37, 38, 41–43, 47] and only three studies used the PCA [27, 28, 34]. Meta-analysis could not be conducted for beta diversity and was not considered as an outcome as the findings were in an accessible format. We tried contacting authors of the papers for raw primary data for beta-diversity but failed to receive any additional information.
Relative abundance of bacterial genera:
One of the main findings of the review was consistency of the identified bacteria among the studies. We tried to identify the most abundant species before and after periodontal intervention and tried to find the common species that decreased and increased after periodontal intervention (Additional file S1). There was more prevalence of bacteria of red complex in pre-intervention stage and more health associated bacteria after intervention. Post intervention Porphyromonas, Fusobacterium, Tannerella, Treponema, Selenomonas, Parvimonas, TM7, Fillibactor, Fretibacterium, and Campylobacter decreased in number. While Escherichia, Neisseria, Prevotella, Capnocytophaga, Lautropia, Haemophilus, Veillonella, Actinomyces, Streptococcus and Rothia increased in number post intervention (Fig. 2).
Synthesis of results:
Alpha diversity compared within test and control groups from baseline to post treatment:
Meta-analysis could not be conducted for studies that did not include mean and SD. The difference in alpha diversity from baseline to post intervention was compared independently for both pooled test and pooled control groups. Post intervention only the pooled test group showed significant changes in richness, Shannon’s diversity and Chao 1 index. Seven trials [10, 11, 28, 29, 32, 43, 44] revealed a decrease in richness in the treatment group (SMD = 0.24; 95% CI= -0.03, 0.46) (Fig. 3). In contrast, the richness resembled baseline in the control group (SMD=-0.35, 95% CI= -0.59, 0.12). Eight studies were meta-analysed for Shannon’s diversity [7, 10, 11, 27–29, 35, 43] (Fig. 4). The test group showed decrease in Shannon diversity in the post treatment samples (SMD: -0.39, 95% CI: 0.20, 0.58), whereas the control group (SMD: -0.24, CI: -0.45, -0.04) resembled baseline. While four trials examined the Chao1 of observed species [10, 11, 35, 43] (Additional File S2), and there was a decrease in the Chao 1 Index between baseline and post treatment (T = SMD: 0.42, 95% CI: 0.09, 0.75) in the test group than the control group (C = SMD: -0.28, 95% CI: -0.14, 0.70).
Risk of Bias Assessment:
The robvis tool was used for visualizing he risk of bias assessment (Additional File S3). The risk of bias for each study was assessed for all domains, as described in the Cochrane Handbook [23]. From the 19studies, the overall risk of bias was high for all but three trials [10, 32, 44]. The main reason for high risk bias was unclear reporting about random sequence generation and allocation concealment.