The Oral Bacterial Microbiome of Interdental Surfaces in Adolescents According to Carious Risk

Abstract

Adolescence is closely associated with a high risk of caries. The identification of specific bacteria in an oral microniche, the interdental space of the molars, according to carious risk can facilitate the prediction of future caries and the anticipation of the progression or stabilization of caries in adolescents. A cross-sectional clinical study according to the bacteriological criteria of interdental healthy adolescents and carious risk factors—low and high—using a real-time polymerase chain reaction technique was conducted. The presence of 26 oral pathogens from the interdental microbiota of 50 adolescents aged 15 to 17 years were qualitatively and quantitatively analyzed. Bacteria known to be cariogenic (Bifidobacterium dentium, Lactobacillus spp., Rothia dentocariosa, Streptococcus cristatus, Streptococcus mutans, Streptococcus salivarius, Streptococcus sobrinus, and Streptococcus wiggsiae) did not present differences in abundance according to carious risk. Periodontal bacteria from the red complex are positively correlated with carious risk. However, only 3 bacteria—S. sobrinus, E corrodens and T. forsythia—presented a significant increase in the highest group. Estimating the risk of caries associated with bacterial factors in interdental sites of molars in adolescents contributes to the better definition of carious risk status, periodicity and intensity of diagnostic, prevention and restorative services.

Full text

microorganisms
Article
The Oral Bacterial Microbiome of Interdental
Surfaces in Adolescents According to Carious Risk
Camille Inquimbert 1,2,†, Denis Bourgeois 1,†, Manuel Bravo 3, Stéphane Viennot 1,
Paul Tramini 2, Juan Carlos Llodra 3, Nicolas Molinari 4, Claude Dussart 1,
Nicolas Giraudeau 2,†and Florence Carrouel 1,*,†
1Laboratory “Systemic Health Care”, EA4129, University Lyon 1, University of Lyon, 69008 Lyon, France
2Department of Public Health, Faculty of Dental Medicine, University of Montpellier,
34090 Montpellier, France
3Department of Preventive and Community Dentistry, Faculty of Odontology, University of Granada,
18010 Granada, Spain
4Service DIM, CHU de Montpellier, UMR 5149 IMAG, University of Montpellier, 34090 Montpellier, France
*Correspondence: florence.carrouel@univ-lyon1.fr; Tel.: +334-7878-5744
†These authors contributed equally to this work.
Received: 16 August 2019; Accepted: 4 September 2019; Published: 5 September 2019
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Abstract:
Adolescence is closely associated with a high risk of caries. The identification of specific
bacteria in an oral microniche, the interdental space of the molars, according to carious risk can
facilitate the prediction of future caries and the anticipation of the progression or stabilization of caries
in adolescents. A cross-sectional clinical study according to the bacteriological criteria of interdental
healthy adolescents and carious risk factors—low and high—using a real-time polymerase chain
reaction technique was conducted. The presence of 26 oral pathogens from the interdental microbiota
of 50 adolescents aged 15 to 17 years were qualitatively and quantitatively analyzed. Bacteria
known to be cariogenic (Bifidobacterium dentium,Lactobacillus spp., Rothia dentocariosa,Streptococcus
cristatus,Streptococcus mutans,Streptococcus salivarius,Streptococcus sobrinus, and Streptococcus wiggsiae)
did not present di↵erences in abundance according to carious risk. Periodontal bacteria from the
red complex are positively correlated with carious risk. However, only 3 bacteria—S. sobrinus,
E corrodens and T. forsythia—presented a significant increase in the highest group. Estimating the risk
of caries associated with bacterial factors in interdental sites of molars in adolescents contributes to
the better definition of carious risk status, periodicity and intensity of diagnostic, prevention and
restorative services.
Keywords: oral microbiome; adolescents; carious risk; interdental microbiota
1. Introduction
In 2017, the three most common causes of the global burden of diseases in the world were oral
disorders (3.47 billion, 95% UI 3.27–3.68), headache disorders (3.07 billion, 95% UI 2.90–3.27), and
tuberculosis, including latent tuberculosis infection (1.93 billion, 95% UI 1.71–2.20) [
1
]. The same year,
the prevalence of caries on permanent teeth was 23,019,992,000 [
1
]. Dental caries associated with
periodontal disease are, together, the most prevalent microbe-mediated human disease worldwide [
2
].
Dental caries is a biofilm-mediated, sugar-driven, multifactorial, dynamic disease that results
in the phasic demineralization and remineralization of dental tissues [
3
]. Dental caries is the result
of dissolution of the tooth mineral by a reduction in pH due to the sustained fermentation of
carbohydrate by bacteria in a local biofilm structure that limits the ability of saliva to wash away or
bu↵er the acidic metabolic products [
4
–
6
]. The biofilm diversity of tooth surfaces is influenced by
Microorganisms 2019,7, 319; doi:10.3390/microorganisms7090319 www.mdpi.com/journal/microorganisms

Microorganisms 2019,7, 319 2 of 24
carbohydrate consumption and a surface’s health status [
7
]. Caries are associated with dysbiosis of the
tooth-colonizing microbiota, characterized by the accumulation of aciduric and acidophilic bacteria [
8
].
The balance between pathological and protective factors influences the initiation and progression of
caries [3].
The risk for developing caries in individuals depends on factors such as the immune system
and oral microbiome, which themselves are a↵ected by environmental and genetic determinants [
9
].
Interdental space is one of the main sites at risk of dental caries. Indeed, due to their anatomical
arrangement and location, the biological interdental space in healthy subjects with an estimated
diameter of 0.6–1.1 mm is an ecological microniche protected externally by the papillary gingiva [
4
,
10
].
Interdental spaces facilitate the structuration and accumulation of oral biofilms [
11
]. The saliva, due to
anatomic accessibility constraints, cannot circulate favorably. Thus, without adequate saliva, the oral
clearance of sugary or acidic foods will be longer, and less urea is available to help raise the plaque
biofilm pH [
12
]. Moreover, accessibility to interdental spaces by conventional methods of individual
prophylaxis is restricted. If toothbrushing is optimal for cleaning occlusal, facial and lingual/palatal
surfaces of teeth, none of the toothbrushing methods is efficient for eliminating the interproximal
supragingival dental plaque or disrupting the biofilm [
13
]. The e↵ectiveness of plaque removal after
brushing has been estimated at approximately 42% [14].
Adolescence is closely associated with a high risk and progression for caries. In total, 67% of
US teens have experienced tooth caries, with untreated decay in 20% [
15
]. The interproximal faces
of the molars are mainly a↵ected [
16
]. The prevalence of caries has been reported to be 39% at the
age of 12, increasing to 72% at the age of 20–21 [
17
]. Enamel lesions on the proximal surfaces among
16-year-olds account for more than 80% of all caries lesions on these surfaces, no matter whether the
caries prevalence in the population is high or low [
18
]. During adolescence, the changes in dietary habits
associated with excessive high carbohydrate consumption, sugary sodas or popular energy drinks,
foods and snacking could contribute to alter the balance of the oral microbiome [
19
,
20
]. Admittedly,
demineralization can be inhibited by salivary components, antibacterial agents, and fluoride or reversed
by remineralization, which requires calcium, phosphate, and fluoride [
21
]. However, in interdental
spaces, the conditions conferred by the topical application of fluoride, a priority in childhood and
adolescence for the remineralization of enamel and brought daily by toothpaste, then conveyed by
saliva, are not met to provide optimal protection. Adolescents0compliance associated with poor oral
hygiene is advanced [
22
]. Oral hygiene and toothbrushing are not always a priority [
23
]. Access to
community prevention interventions for promoting adolescent oral health, i.e., policy, educational
activities, supervised toothbrushing programs, is more complicated because these mainly focus on
early childhood and childhood [24].
Previous studies have focused on the oral microbiota of children with and without tooth decay.
Moreover, most oral microbiology studies are based on pooled samples [
25
], rather than characterizing
potential di↵erences in microbial composition between discrete sites on teeth. Ribeiro and colleagues
have recently studied bacterial diversity in occlusal biofilms and its relationship with the clinical
surface diagnosis and dietary habits of 12-year-old children [
6
]. Our research is the first to target an
oral microniche, which represents the interdental (ID) space, using real-time polymerase chain reaction
(PCR) in order to analyze qualitatively and quantitatively the bacterial composition and to study its
relation with caries risk factors in adolescents.
Better information on changes in community structure—taxonomic identity and abundance—that
evolve in the aggressive ecosystem of a potentially cariogenic biofilm is thus significant for caries risk
assessment and for progress in developing preventive strategies [
6
]. Caries-risk assessment models
currently involve a combination of factors including diet, fluoride exposure, a susceptible host, and
microflora that interplay with a variety of social, cultural, and behavioral factors [
26
]. Our hypothesis
is that the identification of specific bacteria according to the degree of severity of carious risk can
contribute to risk assessment to predict future caries and anticipate caries progression or stabilization
in the adolescent.

Microorganisms 2019,7, 319 3 of 24
This study investigated the changes in biofilm composition of interdental surfaces in posterior
permanent teeth according to carious risk. Data to identify bacteria from the microbiome associated
with caries risk will be emphasized.
The hypotheses are: (i) The quantification of the oral microbiome at the interproximal surface of
the tooth could contribute to the assessment of carious risk and personalized clinical decision-making,
(ii) the qualitative di↵erentiated analysis of the microbiome could help to predict interproximal
adolescence caries development, (iii) the identification of specific bacterial species could help to
develop novel approaches in their diagnosis and management of carious lesions.
2. Materials and Methods
The Microbiota of Interdental Space of Adolescent according to Risk of Caries (MIARC) trial is
a cross-sectional observational clinical study. The workflow of the experiment is described in Figure 1.
Microorganisms 2019, 7, x FOR PEER REVIEW 2 of 25
hypothesis is that the identification of specific bacteria according to the degree of severity of carious
risk can contribute to risk assessment to predict future caries and anticipate caries progression or
stabilization in the adolescent.
This study investigated the changes in biofilm composition of interdental surfaces in posterior
permanent teeth according to carious risk. Data to identify bacteria from the microbiome associated
with caries risk will be emphasized.
The hypotheses are: (i) The quantification of the oral microbiome at the interproximal surface of
the tooth could contribute to the assessment of carious risk and personalized clinical decision-
making, (ii) the qualitative differentiated analysis of the microbiome could help to predict
interproximal adolescence caries development, (iii) the identification of specific bacterial species
could help to develop novel approaches in their diagnosis and management of carious lesions.
2. Materials and Methods
The Microbiota of Interdental Space of Adolescent according to Risk of Caries (MIARC) trial is
a cross-sectional observational clinical study. The workflow of the experiment is described in Figure
1.
Figure 1. Workflow of the experiment. BOIB: Bleeding on Interdental Brushing; DNA:
Deoxyribonucleic acid; ICDAS: International Caries Detection and Assessment System; PCR:
Polymerase Chain Reaction.
CLASSIFICATION OF
SUBJECTS
in two groups with
Caries Risk Assessment
• Biologica l and protect ive
factors (interview)
• Clinical factors (ICDAS and
salivary tests)
Low caries risk
25 adolescents
100
i
n
t
e
r
den
t
a
l
si
t
es
High caries risk
25 adolescents
100 interdental sites
CLINICAL EXAMINATION
1. Interdental sites examination
Interde ntal sites (15- 16; 25-26; 35-3 6; 45-46)
• Determination of interdental diameter
• Interde ntal collecting sample
• BOIB
All other interdental sites
• Determination of interdental diameter
• BOIB
2. Oral examination
• Plaque Index
• Gingival Inde x
MICROBIOLOGICAL ANALYSIS
• Total DNA extraction
• Real-time PCR:
- Quantification of the total load of bacteria
- Quantification of 26 pathogenic species
DATA ANALYSIS
• Statistical analysis
SELECTION OF SUBJECTS
Inclusio n / Exclusion c riteria
50 adolescents
200 interdental sites
Figure 1.
Workflow of the experiment. BOIB: Bleeding on Interdental Brushing; DNA: Deoxyribonucleic
acid; ICDAS: International Caries Detection and Assessment System; PCR: Polymerase Chain Reaction.
2.1. Study Population
Fifty subjects (male and female) were recruited between November and December 2018 from
a pool of first-time volunteers who were referred to the Department of Public Health of the Oral
Medicine Hospital of Montpellier, France. Written informed consent was obtained from all enrolled

Microorganisms 2019,7, 319 4 of 24
subjects in accordance with the Declaration of Helsinki and authorization of the ethics council. The 50
adolescents were selected based on inclusion criteria and carious risk classification to have 25 subjects
in the high caries risk (HCR) group and 25 subjects in the low caries risk (LCR) group.
The inclusion criteria were: (i) Age 15–17 years old, (ii) presence of teeth (15, 16, 25, 26, 35,
36, 45, and 46), (iii) accessibility of the interdental space for the four sites (15–16, 25–26, 35–36, and
45–46) by the interdental brush in each subject, (iv) the presence of at least 22 natural teeth, (v) good
understanding of the French language, (vi) one of the parents accepts the terms of the study and signs
the written informed consent, (vii) adolescent accepts the terms of the study and signs the written
informed consent.
The clinical inclusion criteria for each premolar-molar interdental site were: (i) Accessibility of
the interdental site for the four sites (15–16, 25–26, 35–36, and 45–46) by the interdental brush in each
subject, (ii) no interdental caries or prosthetics restorations, (iii) no interdental diastema, (iv) no clinical
sign of inflammation such as redness, swelling, or bleeding on probing (BOP) after 30 s, (v) no pocket
depth (PD) >3 mm or clinical attachment loss (CAL) >3 mm, and (vi) the subjects were judged to be
free of gingivitis or periodontitis.
The exclusion criteria were: (i) Smokers, (ii) subjects with any other concomitant systemic disease,
(iii) subjects with daily medication, (iv) subjects with an orthodontic appliance, (v) subjects who have
taken antibiotics in the past three months, (vi) subjects regularly using interdental brushes and/or
dental floss and/or mouthwash, and (vii) subjects unable to answer questions and non-cooperative.
2.2. Ethical Approval and Informed Consent
This study was carried out in accordance with the ethical committee of Sud-Est VI Clermont-
Ferrand (Approval number: AU1371) and the National Commission of Informatics and Liberties,
France (2116544 v 0). The National Agency for the Safety of Medicines and Health Products (ANSM)
approved it on February 13, 2017 (ID-RCB ref: 2017-A00425-48). This study was registered with
ClinicalTrials.gov (identification number ID: NCT03700840).
2.3. Classification of Subjects According to Carious Risk
One trained and calibrated dentist, experienced in the clinical indices, realized the clinical examination.
Subjects were classified into two carious risk groups: Low and high risk. In this study, the
classification of subjects according to their carious risk was adapted from the caries-risk assessment
of the American Academy of Pediatric Dentistry that is based on biological, protective and clinical
factors [27].
First, to know the biological and protective factors, the dentist interviewed the patient face-to-face.
Questions referred to socioeconomic status (mother
0
s and father’s occupation based on French
socio-economic classifications), snacks, brushing, regular visits and dental care (Table 1).
Then, a clinical examination was performed to fulfil the questions concerning the clinical factors
(Table 1). The participants were asked to refrain from oral hygiene measures, eating and drinking for
two hours before clinical examination and interdental sampling.
The presence of active caries and interproximal lesions were measured by the International Caries
Detection and Assessment System (ICDAS). This clinical scoring system allows the detection and the
assessment of caries activity. ICDAS was developed for use in clinical research, clinical practice and
for epidemiological purposes. This scoring system can be used on coronal surfaces and root surfaces
and can be applied to enamel caries, dentine caries, non-cavitated lesions (contrary to many systems)
and cavitated lesions. The ICDAS II system has two-digit coding for the detection criteria of primary
coronal caries. The first one is related to the restoration of teeth and has a coding that ranges from 0 to
9. The second digit ranges from 0 to 6 and is used for coding the caries. A compressed air syringe was
used to dry the teeth during the ICDAS assessment [28].
The salivary tests were performed for all subjects using Saliva-Check BUFFER (GC, Sucy-en-Brie,
France). The tests aimed to investigate hydration, salivary consistency, resting saliva pH, stimulated

Microorganisms 2019,7, 319 5 of 24
saliva flow, stimulated saliva pH and saliva bu↵ering capacity. All tests were performed according to
the instructions of the manufacturer to detect low, moderate, or high salivary risk.
The subject was classified as “high carious risk” if at least one “yes” was selected in the column
“high risk”. The subject was classified as “low carious risk” only if no “yes” was selected in the column
“high risk” for the biological and clinical findings, and “yes” was selected for the protective factors.
Table 1. Adolescent caries risk assessment.
High Risk Low Risk
Biological (interview)
•Patient with low socioeconomic status Yes
•
Patient has >3 between-meal sugar-containing snacks or beverages per day
Yes
Protective (interview)
•Patient brushes teeth daily with fluoridated toothpaste Yes
•Patient has regular dental care Yes
Clinical Findings (ICDAS and salivary tests) ICDAS
•Patient had >1 interproximal lesion Yes
•Patient has active white spot lesions or enamel defects Yes
Salivary tests (hydration, salivary consistency, resting saliva pH, stimulated
saliva flow, stimulated saliva pH and saliva bu↵ering capacity)
•Patient has high salivary risk Yes
2.4. Clinical Examination and Interdental Sample Collection
For all subjects, the same four interdental sites (15–16, 25–26, 35–36, and 45–46) were assessed (total
200 sites). The interdental diameter was determined using a dedicated probe—the CURAPROX IAP
calibration probe (Curaden, Kriens, Switzerland). This probe is a graduated conical instrument with
a rounded end. The working portion includes colored bands from the tip to the base corresponding to
interdental brushes (IDB) by increasing diameter. The largest section of each colored band corresponds
to the cleaning efficiency diameter of the respective brush. This made it possible to select the calibrated
IDB (Curaden) appropriate to the diameter of the interdental space. Each previously selected tooth
was isolated using sterile cotton rolls, and the interdental biofilm as removed using this sterile IDB.
For each sample, the IDBs were placed in 1.5-mL sterile microcentrifuge tubes and stored at 4

C for
further laboratory treatment.
The Bleeding on Interdental Brushing Index (BOIB) [
29
] was evaluated on the four interdental
sites (15–16, 25–26, 35–36, and 45–46), as was the bleeding response to the horizontal pressure applied
in the interdental area by a calibrated IDB. After 30 s, bleeding at each gingival unit was recorded
according to the following scale: 0, absence of bleeding after 30 s; and 1, bleeding after 30 s. Then,
interdental diameters and the BOIB were evaluated for all other interdental sites.
Clinical measurements including first the Gingival Index (GI) and, second, the Plaque Index (PI),
were performed on the 4 sides (buccal, lingual/palatal, mesial, distal) of 6 teeth (12, 16, 24, 36, 32, 44)
(Silness–Löe Index) [
30
]. To evaluate the GI, the tissues surrounding each tooth was divided into
4 gingival scoring units: Distal facial papilla, facial margin, mesial facial papilla and lingua gingival
margin. A periodontal probe was used to assess the bleeding potential of the tissues with a score
from 0–3 (0: Absence of inflammation to 3: Severe inflammation). The scores of the four areas of the
tooth were summed and divided by four to obtain the GI for the tooth. The index for the patient was
calculated by summing the indices for all six teeth and dividing by six. Then, the determination of the
PI recorded both soft debris and mineralized deposits on the 6 teeth. Each of the four surfaces of the
teeth gave a score from 0–3. The scores from the four areas of the tooth were added and divided by
four in order to give the plaque index (0: no plaque to 3: abundance of soft matter within the gingival
pocket and/or on the tooth and gingival margin). The index for the patient was obtained by summing
the indices for all six teeth and dividing by six.

Microorganisms 2019,7, 319 6 of 24
2.5. Microbiological Analysis
2.5.1. Total Deoxyribonucleic Acid (DNA) Extraction
Total DNA was isolated from the interdental brushes using the QIAcube HT Plasticware and
Cador Pathogen 96 QIAcube HT Kit (Qiagen, Hilden, Germany), according to the manufacturer’s
guidelines. The elution volume used in this study was 150
µ
L. DNA quality and quantities were
measured using an ultraviolet spectrophotometer at 260 and 280 nm. The DNA sample was considered
pure if the A260/A280 ratio was in the range of 1.8–2 and the A260/A280 ratio was in the range of 2–2.2.
2.5.2. Quantitative Real-Time PCR Assays
Quantitative real-time PCR was carried out for Total Bacterial Count (TB) and for 26 pathogens:
Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf ),
Treponema denticola (Td), Prevotella intermedia (Pi), Parvimonas micra (Pm), Fusobacterium nucleatum
(Fn), Campylobacter rectus (Cr), Eikenella corrodens (Ec), Prevotella nigrescens (Pn), Campylobacter gracilis
(Cg), Capnocytophaga ochracea (Co), Actinomyces odontolyticus (Ao), Veillonella parvula (Vp), Streptococcus
mutans (Smutans), Streptococcus mitis (Smitis), Streptococcus sobrinus (Ssob), Streptococcus salivarius (Ssal),
Streptococcus sanguinis (Ssan), Streptococcus cristatus (Scri), Rothia dentocariosa (Rd), Bifidobacterium
dentium (Bd), Scardovia wiggsiae (Sw), Clostridium cluster IV (ClosIV)(Clostridium leptum subgroup,
includes Faecalibacterium (Fusobacterium)prausnutzii), Clostridium cluster XIVa and XIVb (ClosXIV)
(Clostridium coccoides–Eubacterium rectale subgroup), and Lactobacillus spp. (Lspp).
Simplex quantitative real-time PCR assays were performed in a volume of 10
µ
L composed of
1
⇥
SYBR Premix Ex Taq
™
(Tli RNaseH Plus) (TaKaRa, Shiga, Japan), 2
µ
L of DNA extract and each
primer at 1
µ
M. The bacterial species-specific PCR primers used in this study were provided by Institut
Clinident SAS (Aix-en-Provence, France) and manufactured by Metabion international AG (Planegg,
Germany). The bacterial primers used in this study are derived from sequences already published and
have been adapted to the real-time PCR conditions (Supplementary Table S1).
The assays were performed on the Rotor-Gene Q thermal cycling system (Qiagen, Hilden,
Germany) with the following program: 95

C for 30 s, followed by 40 cycles of 10 s at 95

C, 10 s at the
appropriate annealing temperature (Supplementary Table S1), and 35 s at 72

C. For the total bacterial
load and that of all species, a final melting curve analysis (70–95

C in 1

C steps at 5 s increments)
was performed. Fluorescence signals were measured every cycle at the end of the extension step and
continuously during the melting curve analysis. The resulting data were analyzed using Rotor-Gene Q
Series software (Qiagen, Hilden, Germany).
Serial dilutions of a bacterial standard DNA provided by Institut Clinident SAS (Aix-en-Provence,
France) were used in each reaction as external standards for the absolute quantification of the targeted
bacterial pathogens. The standard bacterial strains used for standard DNA production came from
DSMZ (Germany), CIP Collection of Institut Pasteur or from BCMM/LMG Bacteria Collection: Aa (DSM
No. 8324), Pg (DSM No. 20709), Tf (CIP No. 105220), Td (DSM No. 14222), Pi (DSM No. 20706),
Pm (DSM No. 20468), Fn (DSM No. 20482), Cr (LMG No. 7613), Ec (DSM No. 8340), Pn (DSM No.
13386), Cg (DSM No. 19528), Co (DSM No. 7271), Ao (DSM No. 43760), Vp (CIP No. 60.1), Rd (DSM No.
43762), Bd (DSM20436), Sw (DSM No. 22547), Lspp (CIP No. 102237), Smitis (DSM No. 12643), Smutans
(DSM No. 20523), Ssob (DSM No. 20742), Ssal (DSM No. 20067), Ssan (DSM No. 20068), Scri (DSM No.
8249), ClosIV (DSM 753), and ClosXIV (DSM No. 935).
The pathogenic strains were cultivated on the appropriate selective media. The total number
of cells (number of colony-forming units) was enumerated three times using a Neubauer chamber.
Serial dilutions ranging from 10xE+2 to 10xE+12 cells were utilized, and each of these dilutions was
enumerated in duplicate. The DNA from each of these dilutions was extracted. A standard curve for
each pathogen was generated as a plot between the crossing point (cycle number) and the initial cell
count. The absolute counts of pathogen were determined using these calibration curves [31].
The limit of quantification (LOQ) of the method is summarized in Supplementary Table S1.

Microorganisms 2019,7, 319 7 of 24
2.6. Statistical Analysis
2.6.1. Sample Size
With an alpha error of 5% (2-sided test), a power of 80%, an intraclass correlation coefficient of 0.8,
and a mean di↵erence of bacteria counts between the two caries risk groups of 1,300,000, a total of
200 sites (which means 50 subjects i.e., 25 subjects per caries risk group) was necessary.
2.6.2. Statistical Tests
The statistical analysis consisted of three main steps: Producing descriptive summaries of the
data, modeling the data using a mixed (linear) model and assessing the correlations between bacterial
abundances. Prior to these steps, we transformed the original count data to handle missing data
points, namely, the measurements that fell under the quantification threshold (LOQ) of the quantitative
real-time PCR device. The missing values for a given species were replaced by half of the corresponding
quantification thresholds given in Supplementary Table S1. We performed simulations to ensure
that this simple strategy provided a reasonable estimation of the mean and standard deviation of the
original count distribution. To test for potential e↵ects of gender, interdental space, and the location of
each site, we used a mixed linear model for the log-count abundance of each species at a measured site.
This model includes three categorical variables as fixed e↵ects (gender, mouth location, and interdental
space) and one categorical variable as a random e↵ect (subject). This random e↵ect was introduced
for a subject to model the correlation between the four sites of a given subject. Each coefficient in the
regression was tested against the null hypothesis, which indicates that the coefficient is zero using
a likelihood ratio test, and we reported that p-values less than 0.05, 0.01, and 0.001 were low, medium,
and strong evidence against the null hypothesis, respectively. To perform the correlation analysis, we
used the residuals of the model described above to avoid over-estimating the inter-site correlation
(sites from the same patient are positively correlated, and we observed that fixed e↵ects can also induce
a correlation among sites). The trees associated with the correlation plot were obtained by hierarchical
clustering with complete linkage. The di↵erence between the two groups of caries risk relative to
age, gender, mouth location, interdental space, BOIB, PI and GI were tested with chi-square tests.
Kruskal–Wallis tests were performed to compare the mean counts for the di↵erent bacterial species
relative to each clinical characteristic.
All statistical analyzes and associated plots were performed using the R environment (R Core
Team, 2015), specifically the lme4 package [32], to estimate the mixed model.
3. Results
3.1. Age, Gender, and Clinical Characteristics in the Two Carious Risk Groups
Table 2summarizes the age, the sex, and clinical assessments of the study groups. The two groups
were each composed of 25 subjects. The age, gender and clinical characteristics were similar in the
two groups (Mann–Whitney and chi-square tests). The low caries risk (LCR) group was composed of
18 females and 7 males, whereas the high caries risk (HCR) group was composed of 15 females and
10 males. The mean age was 16.98
±
0.77 years. The mean number of teeth present was 27.6
±
1.1
(excluding wisdom teeth). Missing teeth (1.3%) were due to orthodontic treatment or agenesis (0.95%)
and caries (0.35%). The mean BOIB score in the low risk group was 96.52% and 96.55% in the high-risk
group. No problems due to oral hygiene were observed (fair plaque index 1.0–1.9, gingival index
1.1–2.0). The interdental spaces studied had a diameter between 0.8 and 1.1 mm.
3.2. Quantification of the Total Genome Count and Bacteria Count According to Carious Risk
The mean counts for the total bacterial load and that of the 26 evaluated species in the interdental
biofilm according to carious risk are reported in Figure 2and Table 3. An average of approximately 10
9.4
bacteria was collected in one interdental space for both groups. The quantity of total bacteria and of

Microorganisms 2019,7, 319 8 of 24
bacteria tested was not significantly modified except for T. forsythia,E. corrodens, and S. sobrinus, which
were significantly increased in the HCR group relative to the LCR group. T. forsythia was 26.3 times,
E. corrodens 4.7 times and S. sobrinus 3.3 times higher in the HCR group than in the LCR group.
Table 2. Age, sex, and characteristics of the full mouth of the study group.
High Caries Risk Low Caries Risk p-Value
Subjects
Age (years) 16.08 ±0.81 15.88 ±0.73 0.49
Gender 0.37
Male 10 (40%) 7 (28%)
Female 15 (60%) 18 (72%)
Full mouth
Teeth 27.80 ±0.82 27.48 ±1.23 0.89
BOIB (%) 96.55 ±7.87 96.52 ±5.13 0.52
PI 1.56 ±0.51 1.76 ±0.43 0.22
GI 1.92 ±0.28 1.72 ±0.46 0.22
Interdental space diameter 0.35
0.8 mm 10 (10%) 10 (10%)
0.9 mm 22 (22%) 31 (31%)
1.1 mm 68 (68%) 59 (59%)
The values are mean
±
standard deviation, the numbers and percentages of subjects are indicated. BOIB: Bleeding
On Interdental Brushing, GI: Gingival Index, PI: Plaque Index.
Table 3. Description (mean ±sd) of bacterial counts (log10x+1) in 200 quadrants (n=50 patients ⇥4
quadrants/patient) and comparison according to caries risk.
Variable All (n=200) Caries Risk
High (n=100) Low (n=100) p-Value
TB 9.42 ±0.48 9.43 ±0.47 9.40 ±0.48 0.796
B. dentium 0.76 ±2.20 0.60 ±1.95 0.91 ±2.41 0.522
Lactobacillus spp. 4.54 ±0.67 4.57 ±0.72 4.50 ±0.60 0.591
R. dentocariosa 5.91 ±1.14 5.73 ±1.43 6.08 ±0.70 0.226
S. cristatus 4.41 ±3.02 4.97 ±2.83 3.85 ±3.11 0.096
S. mutans 1.96 ±2.76 1.95 ±2.81 1.97 ±2.72 0.969
S. salivarius 6.90 ±0.70 7.05 ±0.70 6.75 ±0.67 0.068
S. sobrinus 0.30 ±1.19 0.56 ±1.58 0.04 ±0.43 0.049
S. wiggsiae 5.85 ±2.35 6.09 ±1.88 5.59 ±2.72 0.412
A. odontolyticus 3.77 ±1.19 3.97 ±1.18 3.56 ±1.18 0.120
V. parvula 5.05 ±1.36 5.07 ±1.38 5.02 ±1.32 0.881
A. actinomycetemcomitans 0.28 ±1.32 0.55 ±1.83 0.00 ±0.00 0.076
C. ochracea 4.04 ±2.26 4.18 ±2.11 3.89 ±2.40 0.609
E. corrodens 5.84 ±1.27 6.17 ±1.01 5.50 ±1.40 0.006
S. mitis 5.35 ±0.62 5.48 ±0.63 5.22 ±0.58 0.075
S. sanguinis 7.00 ±0.98 7.05 ±1.22 6.94 ±0.65 0.580
C. rectus 5.89 ±1.79 6.00 ±1.71 5.77 ±1.86 0.560
C. gracilis 4.22 ±1.14 4.31 ±0.89 4.12 ±1.34 0.524
F. nucleatum 7.42 ±0.54 7.40 ±0.56 7.43 ±0.50 0.801
P. intermedia 2.85 ±3.43 3.31 ±3.53 2.38 ±3.27 0.153
P. micra 5.05 ±2.50 4.95 ±2.55 5.14 ±2.45 0.741
P. nigrescens 2.12 ±1.99 2.43 ±1.92 1.80 ±2.01 0.131
P. gingivalis 0.34 ±1.47 0.68 ±2.02 0.00 ±0.00 0.082
T. denticola 1.82 ±3.16 2.17 ±3.39 1.45 ±2.88 0.345
T. forsythia 4.95 ±3.20 5.66 ±2.77 4.24 ±3.43 0.046
Clostridium IV 0.43 ±1.25 0.52 ±1.39 0.34 ±1.09 0.441
Clostridium XIV 6.39 ±0.75 6.54 ±0.67 6.23 ±0.79 0.078
The colors refer to (i) the colors of the Socransky complexes for the purple, green, yellow, orange, and red colors,
(ii) cariogenic bacteria for the pink color and (iii) bacteria from the clostridium group for the gray color.

Microorganisms 2019,7, 319 9 of 24
Microorganisms 2019, 7, x FOR PEER REVIEW 2 of 25
Figure 2. Abundance of bacterial species among the interdental sites in the low carious risk and high
carious risk groups. The counts are reported on a log10 scale. Each box represents the first quartile,
median quartile, and third quartile, from bottom to top. The first box on the left (TB) corresponds to
the total bacteria. The colors in boxes refer to (i) the colors of the Socransky complexes for the purple,
green, yellow, orange, and red colors, (ii) cariogenic bacteria for the pink color and (iii) bacteria from
the clostridium group for the gray color. TB, total bacterial load.
Table 4 describes the distribution of pathogens according to sites and subjects. Lactobacillus spp.,
S. salivarius, S. mitis, F. nucleatum, and Clostridium XIV were detected in all subjects and in all
interdental spaces, whatever the carious risk. Other bacteria were not expressed in each interdental
site nor in each subject. For example, P. gingivalis was detected in 11% of HCR subjects and 12% of
interdental sites from HCR subjects, but this bacterium was not detected in LCR subjects. E. corrodens
was observed in all subjects, in 100% of interdental sites from HCR subjects and in 96% of interdental
sites from LCR subjects.
High Carious Risk
Low Carious Risk
Counts (log
10
scale) Counts (log
10
scale)
0
5
10
15
20
25
30
35
40
TB
B.dentium
Lactobacillus spp
R.dentocariosa
S.cristatus
S.mutans
S.salivarius
S.sobrinus
S.wiggsiae
A.odontolyticus
V.parvula
A.actino a
C.ochracea
E.corrodens
S.mitis
S.sanguinis
C.rectus
C.gracilis
F.nucleatum
P.intermedia
P.micros
P.nigrescens
P.gingivalis
T.denticola
T.forsythensis
Clostridium IV
Clostridium XIV
0
5
10
15
20
25
30
35
40
TB
B.dentium
Lactobacillus spp
R.dentocariosa
S.cristatus
S.mutans
S.salivarius
S.sobrinus
S.wiggsiae
A.odontolyticus
V.parvula
A.actino a
C.ochracea
E.corrodens
S.mitis
S.sanguinis
C.rectus
C.gracilis
F.nucleatum
P.intermedia
P.micros
P.nigrescens
P.gingivalis
T.denticola
T.forsythensis
Clostridium IV
Clostridium XIV
Figure 2.
Abundance of bacterial species among the interdental sites in the low carious risk and high
carious risk groups. The counts are reported on a log10 scale. Each box represents the first quartile,
median quartile, and third quartile, from bottom to top. The first box on the left (TB) corresponds to
the total bacteria. The colors in boxes refer to (i) the colors of the Socransky complexes for the purple,
green, yellow, orange, and red colors, (ii) cariogenic bacteria for the pink color and (iii) bacteria from
the clostridium group for the gray color. TB, total bacterial load.
In the HCR group, all species tested were detected. The most abundant species were F. nucleatum
(10
7.4
bacteria in one ID space), S. salivarius (10
7.05
bacteria in one ID space) and S. sanguinis (10
7.05
bacteria in one ID space). The least abundant species were Clostridium IV (10
0.52
bacteria in one ID
space), A. actinomycetencomitans (10
0.55
bacteria in one ID space), and S. sobrinus (10
0.56
bacteria in one
ID space).
In the LCR group, the most abundant species were F. nucleatum (10
7.43
bacteria in one ID
space), S. sanguinis (10
6.94
bacteria in one ID space) and S. salivarius (10
6.75
bacteria in one ID space).
A. actinomycetencomitans and P. gingivalis were not detected.
Table 4describes the distribution of pathogens according to sites and subjects. Lactobacillus spp.,
S. salivarius,S. mitis,F. nucleatum, and Clostridium XIV were detected in all subjects and in all interdental
spaces, whatever the carious risk. Other bacteria were not expressed in each interdental site nor in each
subject. For example, P. gingivalis was detected in 11% of HCR subjects and 12% of interdental sites
from HCR subjects, but this bacterium was not detected in LCR subjects. E. corrodens was observed
in all subjects, in 100% of interdental sites from HCR subjects and in 96% of interdental sites from
LCR subjects.

Microorganisms 2019,7, 319 10 of 24
Table 4. Distribution of the pathogens according to sites and subjects.
All Sex IDB Size
Male Female 0.6 mm 0.7 mm 0.8 mm 0.9 mm 1.1 mm
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
Positive
Sites 1
Positive
Subjects 2
HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR HCR LCR
n100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 22
TB 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 22
Bd 9 13 6 6 1211 8 11 5 5 00000000201024235935
Lspp 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 22
Rd 96 100 24 25 36 28 9 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 64 59 20 22
Scri 77 62 24 22 31 20 10 6 46 42 14 16 00000000634317 24 11 15 54 35 21 19
Smutans 34 36 15 18 6 10 5 6 28 26 10 12 0000 000056345 10 5 9 24 20 8 13
Ssal 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 22
Ssob 12 1 5 1 301091410000 000020204121 6030
Sw 93 82 24 21 34 27 9 7 59 55 15 14 0000000010 9 5 6 21 25 13 16 62 48 16 18
Ao 96 93 25 25 37 27 10 7 59 66 15 18 000000009 10 5 7 20 29 12 18 67 54 21 22
Vp 95 95 25 25 37 25 10 7 58 70 15 18 0000000010 10 5 7 22 29 13 19 63 56 21 22
Aa 903030106020 000000000000 00009030
Co 83 76 23 23 27 22 8 6 56 54 15 17 0000000010 3 5 3 19 28 12 17 54 45 15 19
Ec 100 96 25 25 40 27 10 7 60 69 15 18 0000000010 9 5 6 22 30 13 19 68 57 21 22
Smitis 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 21 31 13 20 68 59 21 22
Ssan 98 100 25 25 39 28 10 7 59 72 15 18 0000000010 10 5 7 21 31 13 20 67 59 21 22
Cr 95 93 25 25 37 26 10 7 58 67 15 18 0000000010 7 5 6 20 30 13 20 65 56 21 22
Cg 99 93 25 24 39 27 10 7 60 66 15 17 0000000010 10 5 7 22 30 13 19 67 53 21 21
Fn 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 26
Pi 48 36 19 18 17 9 7 4 31 27 12 14 00000000 342310 9 7 8 35 23 14 13
Pn 65 47 21 20 17 10 6 5 48 37 15 15 00000000523214 16 9 6 56 29 19 14
Pm 81 84 24 24 32 26 10 7 49 58 14 17 00000000784617 26 11 15 57 50 20 21
Pg 11 0 3 0 4010702000000000 000020109030
Td 30 21 12 8 10 8 5 4 20 13 7 4 000000001010764322 15 10 6
Tf 83 62 24 20 30 14 10 4 53 48 14 16 00000000975516 19 10 12 58 36 20 16
ClosIV 13 9 7 7 4523945400000000 000052528766
ClosXIV 100 100 25 25 40 28 10 7 60 72 15 18 0000000010 10 5 7 22 31 13 20 68 59 21 22
1
Positive sites correspond to the number of sites expressing one pathogenic species or the total bacteria (TB).
2
Positive subjects indicate the number of subjects expressing one pathogenic
species or the total bacteria. The colors refer to (i) the colors of the Socransky complexes for the purple, green, yellow, orange, and red colors, (ii) cariogenic bacteria for the pink color and
(iii) bacteria from the clostridium group for the gray color. Aa: Aggregatibacter actinomycetemcomitans, Ao: Actinomyces odontolyticus, Bd: Bifidobacterium dentium, Cg: Campylobacter gracilis,
ClosIV: Clostridium cluster IV, ClosXIV: Clostridium cluster XIVa and XIVb, Co: Capnocytophaga ochracea, Cr: Campylobacter rectus, Ec: Eikenella corrodens, Fn: Fusobacterium nucleatum, HCR: High
Carious risk group, LCR: Low Carious Risk group, Lspp: Lactobacillus spp, n: total number of sites or subjects tested, Pg: Porphyromonas gingivalis, Pi: Prevotella intermedia, Pm: Parvimonas
micra, Pn: Prevotella nigrescens, Rd: Rothia dentocariosa, Scri: Streptococcus cristatus, Smitis: Streptococcus mitis, Smutans: Streptococcus mutans, Ssal: Streptococcus salivarius, Ssan: Streptococcus
sanguinis, Ssob: Streptococcus sobrinus, Sw: Scardovia wiggsiae, Td: Treponema denticola, Tf: Tannerella forsythia, Vp: Veillonella parvula.

Microorganisms 2019,7, 319 11 of 24
3.3. E↵ects of Gender, Interdental Diameter and BOIB on the Total Genome Count and Bacteria Count
Table 5summarizes the interactions between caries risk and gender, IDB size or BOIB. For the
majority of the pathogens tested, the gender, the interdental diameter and the BOIB had no significant
e↵ect on the bacteria count. However, the sex significantly impacted the quantity of E. corrodens,
P. nigrescens and S. mutans. The IDB size significantly modified the bacteria counts of T. denticola,
P. nigrescens,C. orchracea,V. parvula,B. dentium,S. cristatus,Clostridium IV, and Clostridium XIV. The BOIB
significantly influenced the quantity of T. denticola,C. rectus,E. corrodens,S. salivarius, and S. sanguinis.
Table 5.
Interactions (p-values) between caries risk and selected variables on bacterial counts (log
10
x+
1) in 200 quadrants (n=50 patients ⇥4 quadrants/patient).
Variable p-Values Interaction Caries Risk x...
Sex IDB Size BOIB%
TB 0.835 0.812 0.376
B. dentium 0.246 0.009 10.208
Lactobacillus spp. 0.857 0.332 0.716
R. dentocariosa 0.595 0.492 0.348
S. cristatus 0.253 0.048 10.166
S. mutans 0.008 10.280 0.057
S. salivarius 0.136 0.144 0.023 1
S. sobrinus 0.123 0.103 0.147
S. wiggsiae 0.139 0.666 0.426
A. odontolyticus 0.179 0.331 0.166
V. parvula 0.882 0.012 10.075
A. actinomycetemcomitans 0.203 0.064 0.194
C. ochracea 0.124 <0.001 10.374
E. corrodens 0.020 10.070 0.015 1
S. mitis 0.299 0.362 0.119
S. sanguinis 0.643 0.421 0.034 1
C. rectus 0.809 0.199 <0.001 1
C. gracilis 0.400 0.752 0.605
F. nucleatum 0.853 0.674 0.594
P. intermedia 0.421 0.095 0.186
P. micra 0.940 0.559 0.105
P. nigrescens 0.001 10.024 10.281
P. gingivalis 0.198 0.203 0.209
T. denticola 0.762 <0.001 10.020 1
T. forsythia 0.112 0.130 0.056
Clostridium IV 0.306 0.005 10.473
Clostridium XIV 0.341 0.014 10.062
1
p<0.05. BOIB: Bleeding on Interdental Brushing, IDB: Interdental Brush, TB: Total of bacteria. The colors refer to
(i) the colors of the Socransky complexes for the purple, green, yellow, orange, and red colors, (ii) cariogenic bacteria
for the pink color and (iii) bacteria from the clostridium group for the gray color.
The counts of E. corrodens and P. nigrescens were lower in the LCR group than in the HCR group for
males and females, but the decreases were significant only for females (Figure 3). No significant e↵ect
was observed for S. mutans for males and females. A significant decrease in the counts of B. dentium,
and C. ochracea was observed for IBD of 0.8 mm. For other bacteria tested and other diameters of IDB,
no significant e↵ect was observed.

Microorganisms 2019,7, 319 12 of 24
Microorganisms 2019, 7, x FOR PEER REVIEW 13 of 25
Figure 3. Abundance of bacterial species according to sex and interdental diameter in the low carious
risk group and in the high carious risk group. The counts are reported on a log10 scale. Total counts
from each pathogen were averaged across sites in each subgroup. Error bars represent standard
deviations. This stratified analysis is restricted to those situations where the interaction between caries
risk and sex (A) or caries risk and IDB size (B) on bacterial counts is significant (p < 0.05) (detailed
results not shown). Comparisons: * p < 0.05, by using SUDAAN 7.0 (procedures DESCRIPT and
REGRESS) to account for clustering (multiple sites within the subjects). The colors in boxes refer to (i)
the colors of the Socransky complexes for the purple, green, yellow, orange, and red colors, (ii)
cariogenic bacteria for the pink color and (iii) bacteria from the clostridium group for the gray color.
TB: total bacterial load.
0
2
4
6
8
Counts of pathogen
(log10 scale)
E. corrodens
0
2
4
6
8
Counts of pathogen
(log10 scale)
P. ni g r e s c en s
0
2
4
6
8
Counts of pathogen
(log10 scale)
S. mutans
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
P. ni g r e s c en s
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
T. den tico la
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
C. ochracea
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
V. pa rv ul a
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
S. cristatus
0
2
4
6
8
10
Counts of pathogen
(log10 scale
B. dentium
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
Clostridium IV
0
2
4
6
8
10
Counts of pathogen
(log10 scale)
Clostridium XIV
All Male Female All Male Female All Male Female
All 0.8 mm 0.9 mm 1.1 mm All 0.8 mm 0.9 mm 1.1 mm
All 0.8 mm 0.9 mm 1.1 mm All 0.8 mm 0.9 mm 1.1 mm
All 0.8 mm 0.9 mm 1.1 mm All 0.8 mm 0.9 mm 1.1 mm
A
ll 0.
8
mm 0.
9
mm 1.1 mm
A
ll 0.
8
mm 0.
9
mm 1.1 mm
A
B
High Caries Risk Low Caries Risk
*
*
*
*
Figure 3.
Abundance of bacterial species according to sex and interdental diameter in the low carious
risk group and in the high carious risk group. The counts are reported on a log10 scale. Total counts
from each pathogen were averaged across sites in each subgroup. Error bars represent standard
deviations. This stratified analysis is restricted to those situations where the interaction between caries
risk and sex (
A
) or caries risk and IDB size (
B
) on bacterial counts is significant (p<0.05) (detailed results
not shown). Comparisons: * p<0.05, by using SUDAAN 7.0 (procedures DESCRIPT and REGRESS) to
account for clustering (multiple sites within the subjects). The colors in boxes refer to (i) the colors of
the Socransky complexes for the purple, green, yellow, orange, and red colors, (ii) cariogenic bacteria
for the pink color and (iii) bacteria from the clostridium group for the gray color. TB: total bacterial load.

Microorganisms 2019,7, 319 13 of 24
Table 6details the e↵ect of BOIB on bacterial count according to caries risk. The quantity of
S. salivarius and S. sanguinis significantly increased in the HCR group, whereas no significant e↵ect was
observed for the LCR group. E. corrodens was not significantly correlated with the BOIB. T. denticola
was significantly increased with the BOIB in the LCR group but not in the HCR group. C. rectus was
correlated with the increase in BOIB in both groups.
Table 6.
Stratified analysis of the e↵ect of BOIB on bacterial counts
1
, according to caries risk level in
200 quadrants (n=50 patients x 4 quadrants/patient).
Variable Caries Risk
High (n=100) Low (n=100)
Streptococcus salivarius r =0.22, p=0.011 r=0.21, p=0.198
Eikenella corrodens r =0.07, p=0.388 r=0.22, p=0.164
Streptococcus sanguinis r =0.16, p=0.008 r=0.23, p=0.213
Campylobacter rectus r =0.36, p<0.001 r=0.35, p=0.030
Treponema denticola r =0.12, p=0.138 r=0.23, p=0.035
1
This stratified analysis is restricted to those situations where the interaction between caries risk and sex (or IDB
size) on bacterial counts is significant (p<0.05) (detailed results not shown). The colors refer to (i) the colors of the
Socransky complexes for the purple, green, yellow, orange, and red colors, (ii) cariogenic bacteria for the pink color
and (iii) bacteria from the clostridium group for the gray color.
3.4. Pathogen Correlations According to Carious Risk
The dendrogram (Figure 4) underscores the correlations between the 26 pathogenic species and
the ID sites for the HCR group and the LCR group. Even after the removal of the fixed e↵ects related
to interdental space and age, and the subtraction of the inter-site correlations, the matrix still revealed
a strong correlation structure, which appeared as two groups (or clusters) of correlated species for the
HCR group and one for the LCR group.
Microorganisms 2019, 7, x FOR PEER REVIEW 15 of 25
Figure 4. Correlation plot of the abundances of the bacterial species, corrected for age, interdental
space and individual-specific effects. (A) High caries risk group, (B) low caries risk group. Yellow
indicates positive correlations, whereas red indicates the absence of correlations. The colored leaves
on the top dendrogram represent (i) the colors of the Socransky complexes for the purple, green,
yellow, orange, and red colors, (ii) cariogenic bacteria for the pink color and (iii) bacteria from the
clostridium group for the gray color.
4. Discussion
This study provides a comprehensive survey of the interdental microbiota in adolescents, a
target group that remains poorly explored, according to carious risk. To our knowledge, there is no
scientific reference in the literature that jointly targets a cross-sectional clinical study (MIARC),
according to the bacteriological criteria of interdental healthy adolescents, caries risk factors and the
use of a real-time PCR technique. A few studies focused on the carious microbiota of adolescents, but
these studies were concerned with the subgingival microbiota without specific location, during
and/or after orthodontic treatment [33,34], and quantified few bacteria (A. actinomycetemcomitans, P.
gingivalis, P. intermedia, T. forsythia) [35].
Most research investigating the commensal oral microbiome are focused on disease or are
restricted in methodology. To diagnose and treat caries at an early and reversible stage, an in-depth
definition of health is indispensable [36]. Our research option consisted of carrying out real-time PCR
analysis of the interdental microbiota of caries-free sites in healthy adolescents with or without a
carious risk. All pathogens considered in our study have been before identified in oral samples of
children, adolescents, or young adults [37–40]. Our study did not only focus on cariogenic bacteria
because the classification of oral bacteria seems more complex. Effectively, some studies
demonstrated a positive association between periodontitis and caries, whereas others demonstrated
a negative association [41–43]. These studies observed clinical signs, and only one study considered
the microbiota, but the subjects were clinically affected by these oral diseases [44]. Qualitatively and
quantitatively, the presence of 26 oral pathogens was analyzed. Among them were bacteria
commonly classified as cariogenic bacteria—B. dentium, Lactobacillus spp., R. dentocariosa, S. cristatus,
S. mutans, S. salivarius, S. sobrinus, S. wiggsiae; bacteria considered periodontopathogenic—bacteria
from the purple complex (A. odontolyticus, V. parvula), the green complex (A. actinomycetemcomitan, C.
orchrocea, E. corrodens), the yellow complex (S. mitis, S. sanguinis), the orange complex (C. rectus, C.
S.cristatus
C.ochracea
B.dentium
P.nigrescens
P.intermedia
C.gracilis
S.sobrinus
ClostridiumIV
T.forsythensis
S.mutans
S.wiggsiae
E.corrodens
V.parvula
Lactobacillus spp
R.dentocariosa
S.mitis
A.odontolyticus
S.salivarius
S.sanguinis
F.nucleatum
ClostridiumXIV
P.micra
T.denticola
Total Bacteria
C.rectus
C.gracilis
P.micra
C.rectus
P.gingivalis
A. actinomycetemcomitans
V.parvula
F.nucleatum
R.dentocariosa
S.cristatus
T.denticola
T.forsythensis
C.orchracea
P.nigrescens
B.dentium
ClostridiumIV
S.sobrinus
P.intermedia
S.mutans
S.wiggsiae
E.corrodens
Lactobacillus spp
Total Bacteria
ClostridiumXIV
S.mitis
S.sanguinis
A.odontolyticus
S.salivarius
AB
S.cristatus
C.ochracea
B.dentium
P.nigrescens
P.intermedia
C.gracilis
S.sobrinus
ClostridiumIV
T.forsythensis
S.mutans
S.wiggsiae
E.corrodens
V.parvula
Lactobacillus spp
R.dentocariosa
S.mitis
A.odontolyticus
S.salivarius
S.sanguinis
F.nucleatum
ClostridiumXIV
P.micra
T.denticola
Total Bacteria
C.rectus
C.gracilis
P.micra
C.rectus
P.gingivalis
A. actinomycetemcomitans
V.parvula
F.nucleatum
R.dentocariosa
S.cristatus
T.denticola
T.forsythensis
C.orchracea
P.nigrescens
B.dentium
ClostridiumIV
S.sobrinus
P.intermedia
S.mutans
S.wiggsiae
E.corrodens
Lactobacillus spp
Total Bacteria
ClostridiumXIV
S.mitis
S.sanguinis
A.odontolyticus
S.salivarius
Figure 4.
Correlation plot of the abundances of the bacterial species, corrected for age, interdental
space and individual-specific e↵ects. (
A
) High caries risk group, (
B
) low caries risk group. Yellow
indicates positive correlations, whereas red indicates the absence of correlations. The colored leaves on
the top dendrogram represent (i) the colors of the Socransky complexes for the purple, green, yellow,
orange, and red colors, (ii) cariogenic bacteria for the pink color and (iii) bacteria from the clostridium
group for the gray color.

Microorganisms 2019,7, 319 14 of 24
The first cluster of the HCR group was composed of C. gracilis,P. micra,C. rectus,P. gingivalis,
A. actinomycetemcomitans,V. parvula, and F. nucleatum, whereas the second cluster was composed of
Lactobacillus spp., Clostridium XIV,S. mitis,S. sanguinis,A. odontolyticus, and S. salivarius.
The cluster from the LCR group was composed of S. mitis,A. odontolyticus,S. salivarius,S. sanguinis,
F. nucleatum,Clostridium XIV,P. micra,T. denticola, and C. rectus.
4. Discussion
This study provides a comprehensive survey of the interdental microbiota in adolescents, a target
group that remains poorly explored, according to carious risk. To our knowledge, there is no scientific
reference in the literature that jointly targets a cross-sectional clinical study (MIARC), according to the
bacteriological criteria of interdental healthy adolescents, caries risk factors and the use of a real-time
PCR technique. A few studies focused on the carious microbiota of adolescents, but these studies were
concerned with the subgingival microbiota without specific location, during and/or after orthodontic
treatment [
33
,
34
], and quantified few bacteria (A. actinomycetemcomitans,P. gingivalis,P. intermedia,
T. forsythia)[35].
Most research investigating the commensal oral microbiome are focused on disease or are
restricted in methodology. To diagnose and treat caries at an early and reversible stage, an in-depth
definition of health is indispensable [
36
]. Our research option consisted of carrying out real-time
PCR analysis of the interdental microbiota of caries-free sites in healthy adolescents with or without
a carious risk. All pathogens considered in our study have been before identified in oral samples
of children, adolescents, or young adults [
37
–
40
]. Our study did not only focus on cariogenic
bacteria because the classification of oral bacteria seems more complex. E↵ectively, some studies
demonstrated a positive association between periodontitis and caries, whereas others demonstrated
a negative association [
41
–
43
]. These studies observed clinical signs, and only one study considered
the microbiota, but the subjects were clinically a↵ected by these oral diseases [
44
]. Qualitatively and
quantitatively, the presence of 26 oral pathogens was analyzed. Among them were bacteria commonly
classified as cariogenic bacteria—B. dentium,Lactobacillus spp., R. dentocariosa,S. cristatus,S. mutans,
S. salivarius,S. sobrinus,S. wiggsiae; bacteria considered periodontopathogenic—bacteria from the
purple complex (A. odontolyticus,V. parvula), the green complex (A. actinomycetemcomitan,C. orchrocea,
E. corrodens), the yellow complex (S. mitis,S. sanguinis), the orange complex (C. rectus,C. gracilis,
F. nucleatum,P. intermedia,P. micra,P. nigrescens), the red complex (P. gingivalis,T. denticola,T. forsythia);
and others such as Clostridium IV and Clostridium XIV.
A particular focus of our study is a Caries Risk Assessment (CRA) system. Indeed, the carious
lesion is a multifactorial disease principally due to carious biofilm and sugar consumption. Caries-risk
assessment models currently involve a combination of factors including diet, fluoride exposure,
a susceptible host, and microflora that interplay with a variety of social, cultural, and behavioral
factors [
26
]. The initiation of dental caries results from the balance between risk and protective factors.
This interplay between factors underpins the classification of individuals and groups into caries risk
categories, allowing an increasingly tailored approach to care. The classification criteria for CRA
systems were determined by combining scientific evidence and expert opinion. From these CRAs,
practitioners analyze the various clinical and social factors of a patient and can thus assign a carious
risk status [
45
]. For low-risk patients, there is no necessity for further preventive professional treatment,
and they should be o↵ered an extended follow-up [
46
]. For high-risk patients, preventive actions must
be taken to reduce the incidence and severity of future carious lesions [
46
]. This individual scheduling
of preventive and follow-up activity better appropriates the use of dental resources and lowers dental
costs for certain individuals [
46
]. At present, the criteria needed to perform quantitative caries risk
assessment evaluations are still incomplete [26].
Few CRA classifications consider the quantification of bacteria, and generally such bacteria are
singular and unique. In these few CRA classifications, the quantification of S. mutans in the saliva has
been considered [
47
]. However, the results concerning the link between S. mutans and the development

Microorganisms 2019,7, 319 15 of 24
of dental caries is not clear. Some studies demonstrated a real association between S. mutans and the
carious lesion, whereas others revealed no clear association [
48
]. Moreover, the amount of S. mutans
needed to initiate the carious lesion varies according to the study [
49
]. Thus, to use this bacterium or
other bacteria as a biomarker in a CRA classification, it is necessary to identify specific biomarkers
associated with carious lesions and to normalize their quantification.
Concerning bacteria classified as cariogenic bacteria (B. dentium,Lactobacillus spp., R. dentocariosa,
S. cristatus,S. mutans,S. salivarius,S. sobrinus,S. wiggsiae), no significant di↵erence was observed in the
HCR subjects relative to the LCR subjects. Therefore, these bacteria do not represent good predictive
factors for CRA. Cariogenic bacteria are acidogenic and acid-tolerant species [50].
For many years, S. mutans was considered the major oral pathogen implicated in the initiation of
caries. However, this role has been questioned [
51
]. E↵ectively, caries have been observed without the
presence of S. mutans [
50
,
52
–
54
]. In our study, S. mutans was expressed in small quantities (10
1.97
for
the LCR and 10
1.95
for the HCR group). Our results confirmed that S. mutans alone could not be used to
predict carious risk as previously demonstrated by Gross and colleagues [
50
]. A CRA classification that
uses the quantification of S. mutans in the saliva must be used with care [
21
]. In some studies, caries
was associated with the absence of S. mutans and the presence of Lactobacillus [
55
,
56
], B. dentium [
55
],
and S. wiggsiae [57].
S. sobrinus, closely related to S. mutans, is described as a major bacterium in the apparition of
carious lesions [
58
]. This pathogen was significantly increased by three times in the HCR group relative
to the LCR group, and it could also be a predictive marker for caries, as described by Gross and
colleagues for the supragingival biofilm [
50
]. However, contrary to this previous study, this pathogen
was expressed only in 20% of the subjects from the HCR group, which limits its detection and its
potential to be an interesting predictive marker of caries.
S. salivarius has been associated with caries due to its high cariogenic capacity [
55
]. Moreover,
in a rat model, this pathogen was able to induce caries. Contrary to these results, in our study, this
pathogen was expressed in all subjects at equal levels in both groups [
59
]. These divergent results
could be explained by the fact that some S. salivarius strains such as S. salivarius M18 have probiotic
activity and help to fight against cariogenic bacteria [60].
S. wiggsiae is classified as cariogenic because it is able to tolerate acid and to produce acid from
several sugars at low pH [
65
]. This pathogen was significantly associated with severe-early childhood
caries and was also observed in adolescents presenting initial carious lesions with fixed orthodontic
appliances [
61
,
62
]. Contrary to these studies, our results indicated that S. wiggsiae was highly expressed
whatever the carious risk and in more than 84% of subjects in both groups.
S. cristatus is considered an important cariogenic species due to its association with childhood
caries [
57
,
63
]. Our results are in accordance with those of Dzidic and colleagues because, the quantity
of S. cristatus is 13 times higher in HCR subjects but not significantly [
63
]. This pathogen is known
to be inversely correlated to the presence of P. gingivalis [
64
]. Our results are in accordance with this
because P. gingivalis represented 10
0.35
counts in one ID space, whereas S. cristatus represented 10
4.42
counts in one ID space.
The relationship between Lactobacillus and caries is well established [
65
,
66
]. Lactobacillus
spp. produces water-insoluble polysaccharides that promote bacterial attachment to the tooth
surface. Therefore, other bacteria are able to fix the organic acids that are confined that modify the
microenvironment to enrich the aciduric microflora [
67
]. Although lactobacilli play an important role in
the prognosis of caries, they are unlikely to play an important role in the development of caries [
68
,
69
].
Therefore, the number of salivary lactobacilli may be indirectly related to the progression of caries. Our
study revealed that Lactobacillus spp. was present in all caries-free subjects, but the rate was higher in
the HCR group, although not significant.
B. dentium was isolated from the oral biofilm and from saliva [
70
]. Bifidobacterium was isolated from
80% of plaque samples from early childhood caries patients [
55
,
71
] and from the saliva of children with
a higher frequency in caries-a↵ected children than in caries-free children [
72
]. B. dentium was defined

Microorganisms 2019,7, 319 16 of 24
as a novel caries-associated bacterium with an acidogenic potential and a high fluoride tolerance [
73
].
Our results revealed that this pathogen is only in 24% of the subjects in the HCR and LCR groups, and
no significant di↵erence was observed between the two groups. Therefore, this bacterium cannot be
used for CRA.
R. dentocariosa was identified as a cariogenic bacteria [
74
,
75
]. Jiang and colleagues observed that
the quantity of this pathogen was higher in the saliva from children with caries than caries-free children.
They suggested that it could be a predictive marker for CRA. However, our results indicated that
R. dentocariosa was expressed in 96% of subjects from the HCR group and 100% of subjects from the
LCR group with no significant di↵erence in quantity between the two groups. Therefore, this pathogen
alone cannot be used for CRA.
Studies agree that oral diseases are not only attributable to specific bacteria but that they result
from dysbiosis of the oral microbiota. The apparition of oral disease could be due to the decrease of
bacterial taxon and to the presence of key pathogens [76,77].
Concerning bacteria classified as periodontal bacteria, as previously described, bacteria from
the blue, yellow, green, and purple complexes of Socransky are associated with oral health, whereas
bacteria from the orange and red complexes are associated with dysbiosis of the microbiota and the
apparition of gingivitis and, later, periodontitis [
78
]. Our study reveals that bacteria associated with
Socransky complexes and thus with gingivitis and periodontitis are present in adolescents that present
no signs of gingivitis. The higher quantity of bacteria from the red complex in the HCR group than the
LCR group indicated a positive correlation between periodonthopathogenic bacteria and carious risk.
Previously, Durand and colleagues demonstrated a positive correlation between periodontal disease
severity and the decayed, missing, filled teeth surfaces index [44].
Concerning the red complex, our results indicated that P. gingivalis was not detected in the subjects
of the LCR group and was only detected in 11% of the interdental sites in the HCR group. This
could be explained because in both groups the quantity of S. mutans (10
1.97
in the LCR group and
101.95 in the HCR group) and S. sanguinis (106.94 in the LCR group and 107.05 in the HCR group) were
high compared with quantity of P. gingivalis (not detected in the LCR group and 10
0.68
in the HCR
group). A previous study demonstrated that the growth of P. gingivalis was significantly inhibited
by supernatants from S. mutans and S. sanguinis [
79
]. Moreover, S. cristatus (10
4.97
in the HCR group)
could also inhibit the growth of P. gingivalis because S. cristatus is able to inhibit the expression of
virulence genes of P. gingivalis and consequently delay the growth of dental plaques [80].
T. denticola was detected in low quantities (10
2.17
in the HCR group) and was higher in the HCR
group than the LCR group. This bacterium was detected in interdental sites expressing bacteria from
the red or the orange complex. This is in accordance with previous studies that demonstrated that
T. denticola is unable to adhere on oral surfaces as it is only able to colonize to form oral biofilms when
other periodontal bacteria are present. In subgingival dental biofilms, T. denticola is typically associated
with P. gingivalis [
81
]. A chymotrypsin-like proteinase found within a high-molecular-mass complex
on the cell surface of T. denticola mediates its adherence to other potential periodontal pathogens such
as P. gingivalis, F. nucleatum, P. intermedia and P. micra [82].
T. forsythia was expressed in 96% of adolescents (83% of interdental sites) from the HCR group and
was the only bacteria from the red complex that significantly increased in the HCR group relative to the
LCR group. This bacterium could represent an interesting biomarker for the CRA system. However,
as it was expressed in 93% of subjects and 83% of interdental spaces from the HCR group, some false
negatives could appear. T. forsythia acts with P. gingivalis to initiate chronic periodontitis and was
not described in carious lesions. In our study, T. forsythia was not correlated with P. gingivalis and so
could act with other bacteria such as S. cristatus and R. dentocariosa, which are known to be implicated
in caries [
74
,
83
]. Moreover, T. forsythia was correlated with T. denticola, confirming previous results
demonstrating that T. denticola and T. forsythia are involved in protein–protein interactions through
a leucine-rich repeat in proteins [84].

Microorganisms 2019,7, 319 17 of 24
Bacteria from the orange complex do not seem to have a key role in carious risk because no
significant di↵erence was observed by comparing the HCR and the LCR groups. In fact, these bacteria
have a key role in the initiation of subgingival microbiota dysbiosis [
85
], but no role in carious lesions
was described.
P. nigrescens was previously detected in children caries [
86
], which is in accordance with our results
indicating that this pathogen was detected in 76% of subjects from the HCR group. For this pathogen,
sex significantly impacted carious risk. A higher quantity was detected in females than males and
in the female HCR group relative to the female LCR group. Nakagawa and colleagues previously
demonstrated that P. nigrescens was significantly increased in puberty compared with prepuberty in
females but not in males. This could be associated with sex hormone modifications [87].
Contrary to the study of Gross and colleagues that demonstrated that C. rectus decreased as caries
progressed, our study revealed no modification according to caries risk that could be explained because
those authors sampled supragingival biofilm from carious lesions, whereas we analyzed interdental
biofilms associated with a healthy surface [
56
]. However, our study revealed that C. rectus increased
significantly in the HCR group with the BOIB, suggesting that this pathogen could also be associated
with interdental inflammation. Indeed, C. rectus is known to be present in chronic gingivitis [88].
From the yellow complex, S. mitis and S. sanguinis were not significantly modified when the HCR
and the LCR group were compared. This result aligns with those from previous studies indicating
that S. sanguinis and S. mitis are significantly associated with dental health [
89
–
91
]. S. sanguinis is
known to be a pioneer colonizer that permits the adhesion of other microorganisms and biofilm
formation [
50
,
92
,
93
]. Our results indicated that the quantity of S. sanguinis is 90 times higher than that
of S. mutans. This result is in accordance with the study of Caufield and colleagues, who demonstrated
a higher level of S. sanguinis relative to the level of S. mutans in the saliva [94].
Of the green complex bacteria studied, only E. corrodens was significantly increased with carious
risk. Moreover, this pathogen was expressed in each subject and in each interdental space from
the HCR group and in 96% of subjects from the LCR group. These results are in accordance with
those from Choi and colleagues, who demonstrated that this pathogen was present in the saliva from
periodontally healthy subjects [
95
]. Previous studies have demonstrated that E. corrodens is important
for the progression of periodontal disease in young subjects [
95
,
96
]. This bacterium participates in
the early stage of biofilm formation by mediating the specific co-aggregation of bacteria from the oral
cavity. It is a middle colonizer, linking early colonizers such as Streptococci and late colonizers such
as P. gingivalis [
97
]. As our results indicated that this pathogen could be a key factor for the carious
process and that it was phylogenetically close to the cluster (Lactobacillus spp., Clostridium XIV,S. mitis,
S. sanguinis,A. odontolyticus, and S. salivarius) from the HCR group, it could participate, as previously
described in periodontitis, in biofilm formation and could permit cariogenic bacteria to adhere to
biofilm. In our study, females from the HCR group had a higher quantity of E. corrodens than females
from the LCR group. This could be explained because females have higher caries prevalence than
males [
98
–
101
]. Moreover, E. corrodens is associated with systemic diseases such as spinal, head, and
neck infection or endocarditis [
102
–
106
]. Therefore, it could be a predictive marker of carious disease
but also of systemic diseases.
Concerning the purple complex, V. parvula and A. odontolyticus were not linked with carious
risk but were expressed in more than 90% of the interdental spaces tested. Groos and colleagues
demonstrated that V. parvula increased with the carious lesion step [
50
], and Kanasi and colleagues
identified it in carious lesions from children [
107
]. V. parvula is important in biofilm formation. It can
co-aggregate with other microorganisms such as S. mutans. Indeed, V. parvula cannot fix itself on the
surface of teeth and attaches to S. mutans [
108
]. A. odontolyticus also acts in the early formation of
biofilm [109]. A. odontolyticus was previously detected in carious lesions [86,110,111].
A recent study demonstrated that Clostridium was positively and significantly correlated to caries
and pigment in primary dentition [
112
]. However, even in our study, all subjects expressed Clostridium
XIV, while only 13% of the HCR group and 9% of the LCR group expressed Clostridium IV, and no

Microorganisms 2019,7, 319 18 of 24
significant di↵erences were observed between the HCR and LCR groups. However, Clostridium XIV
was 2 times higher in the HCR than in the LCR group, which could be in accordance with the study by
Li and colleagues [112].
The human oral microbiota has recently become a new focus for its involvement in systemic
diseases [
113
,
114
]. Our work underlines the presence in the interdental niche of healthy adolescents
of a number of oral bacteria described in the literature for their association with systemic diseases.
E↵ectively, B. dentium is known to cause bacteraemia [
115
]. A. odontolyticus has been identified in
actinomycosis throughout the body [
116
,
117
]. R. dentocariosa and S. mutans are known to be associated
with bacteraemia and infective endocarditis [
118
,
119
]. E. corrodens has been described in cardiovascular
diseases (CVD) [
120
,
121
], and C. ochracea has also been described in lupus [
122
,
123
]. F. nucleatum has
been related to CVD, Alzheimer
0
s, and adverse pregnancy [
124
]. P. gingivalis was linked to CVD,
respiratory tract infection, cancer, Alzheimer
0
s, rheumatoid arthritis, and adverse pregnancy [
124
,
125
].
CVD and Alzheimer
0
s were also associated with P. intermedia,T. denticola, and T. forsythia, which
was also implicated in respiratory tract infection [
124
]. C. rectus and V. parvula were linked to
CDV [
124
]. P. nigrescens was discovered in rheumatoid arthritis [
125
]. Therefore, the accumulation
of these pathogenic bacteria in the interdental space must be considered. These bacteria represent
a risk-predisposing factor to the development of systemic diseases in the future. As interdental hygiene
is often neglected (the toothbrush is not used enough), it is necessary to introduce interdental brushing
as early as adolescence.
5. Conclusions
Estimating the risk of caries associated with bacterial factors in interproximal sites in adolescents
will permit a more evidence-based strategy for medical referrals for certain individuals and contribute
to a better definition of carious risk status, periodicity and intensity of diagnostic, prevention and
restorative services. In our study, the analysis of the interdental microbiota from a sample of
adolescents di↵erentiated by level of caries risk highlights the potential role that three bacteria
(S. sobrinus,E. corrodens, and T. forsythia) could have on the predictive development of interproximal
caries. E. corrodens appears to be more interesting because it was found in all interdental sites from
the HCR group and, it had the lowest p-value when comparing HCR and LCR groups. Moreover,
the detection of S. sobrinus and T. forsythia could give false negatives because they were not detected
in all the interdental sites and all the adolescents for the HCR group. While existing tools for caries
risk assessment and interproximal adolescence caries prediction have restricted manageable clinical
value, introducing a biological, non-subjective and personalized marker could be recommended to
increase the CRA classification. Sensitive assessment of metabolic process using microbial biomarkers
at the biofilm–enamel interface could thus perform it possible to specify endpoints prior to the clinical
expression. The knowledge obtained from our trial contributes to generating active hypotheses related
to the composition and variability of the oral microbiome in the interdental space of adolescents.
These hypotheses can be prospectively explored in longitudinal cohort studies, using next generation
sequencing techniques, essential to identify other biomarkers of the disease in real time.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2076-2607/7/9/319/s1,
Table S1: Species-specific and ubiquitous real-time PCR primers for 26 bacteria, annealing temperatures, and the
limits of quantification.
Author Contributions:
Conceptualization, D.B. and F.C.; methodology, F.C. and C.I.; validation, C.I., S.V. and P.T.;
formal analysis, M.B., P.T. and N.M.; investigation, C.I.; writing—original draft preparation, D.B., F.C. and C.I.;
writing—review and editing, D.B., M.B., F.C., C.D., N.G., C.I., J.C.L., N.M., P.T., S.V.; visualization, F.C., M.B., C.I.,
N.M., P.T.; project administration, F.C. and P.T.
Funding: This research received no external funding.
Acknowledgments:
We acknowledge the support of our work by Institut Clinident sas (Aix-en-Provence, France)
and all the participants.
Conflicts of Interest: The authors declare no conflict of interest.

Microorganisms 2019,7, 319 19 of 24
References
1.
Global Burden of Disease Study 2017 (GBD 2017) Data Resources|GHDx. Available online: http://ghdx.
healthdata.org/gbd-2017 (accessed on 14 December 2018).
2.
Valm, A.M. The structure of dental plaque microbial communities in the transition from health to dental
caries and periodontal disease. J. Mol. Biol. 2019,431, 2957–2969. [CrossRef]
3.
Pitts, N.B.; Zero, D.T.; Marsh, P.D.; Ekstrand, K.; Weintraub, J.A.; Ramos-Gomez, F.; Tagami, J.; Twetman, S.;
Tsakos, G.; Ismail, A. Dental caries. Nat. Rev. Dis. Primer 2017,3, 17030. [CrossRef]
4.
Abou Neel, E.A.; Aljabo, A.; Strange, A.; Ibrahim, S.; Coathup, M.; Young, A.M.; Bozec, L.; Mudera, V.
Demineralization–remineralization dynamics in teeth and bone. Int. J. Nanomed.
2016
,11, 4743–4763.
[CrossRef]
5.
Chenicheri, S.R.U.; Ramachandran, R.; Thomas, V.; Wood, A. Insight into oral biofilm: Primary, secondary
and residual caries and phyto-challenged solutions. Open Dent. J. 2017,11.[CrossRef]
6.
Ribeiro, A.A.; Azcarate-Peril, M.A.; Cadenas, M.B.; Butz, N.; Paster, B.J.; Chen, T.; Bair, E.; Arnold, R.R. The
oral bacterial microbiome of occlusal surfaces in children and its association with diet and caries. PLoS ONE
2017,12.[CrossRef]
7.
Zaura, E.; Brandt, B.W.; Prodan, A.; Teixeira de Mattos, M.J.; Imangaliyev, S.; Kool, J.; Buijs, M.J.; Jagers, F.L.;
Hennequin-Hoenderdos, N.L.; Slot, D.E.; et al. On the ecosystemic network of saliva in healthy young adults.
ISME J. 2017,11, 1218–1231. [CrossRef]
8.
Takahashi, N.; Nyvad, B. The role of bacteria in the caries process: Ecological perspectives. J. Dent. Res.
2011
,
90, 294–303. [CrossRef]
9.
Mosaddad; Tahmasebi, E.; Yazdanian, A.; Rezvani, M.B.; Seifalian, A.; Yazdanian, M.; Tebyanian, H. Oral
microbial biofilms: An update. Eur. J. Clin. Microbiol. Infect. Dis. 2019.[CrossRef]
10.
Carrouel, F.; Llodra, J.C.; Viennot, S.; Santamaria, J.; Bravo, M.; Bourgeois, D. Access to interdental brushing
in periodontal healthy young adults: A cross-sectional Study. PLoS ONE 2016,11, e0155467. [CrossRef]
11.
Ng, E.; Lim, L.P. An Overview of di↵erent interdental cleaning aids and their e↵ectiveness. Dent. J.
2019
,7,
56. [CrossRef]
12.
Maheswari, S.U.; Raja, J.; Kumar, A.; Seelan, R.G. Caries management by risk assessment: A review on
current strategies for caries prevention and management. J. Pharm. Bioallied Sci. 2015,7, S320–S324.
13.
Worthington, H.V.; MacDonald, L.; Poklepovic Pericic, T.; Sambunjak, D.; Johnson, T.M.; Imai, P.; Clarkson, J.E.
Home use of interdental cleaning devices, in addition to toothbrushing, for preventing and controlling
periodontal diseases and dental caries. Cochrane Database Syst. Rev. 2019,4, CD012018. [CrossRef]
14.
Slot, D.E.; Wiggelinkhuizen, L.; Rosema, N.A.M.; Van der Weijden, G.A. The efficacy of manual toothbrushes
following a brushing exercise: A systematic review. Int. J. Dent. Hyg. 2012,10, 187–197. [CrossRef]
15.
Dye, B.A.; Thornton-Evans, G.; Li, X.; Iafolla, T.J. Dental caries and sealant prevalence in children and
adolescents in the United States, 2011–2012. NCHS Data Brief 2015, 1–8.
16.
Schwendicke, F.; Paris, S.; Stolpe, M. Detection and treatment of proximal caries lesions: Milieu-specific
cost-e↵ectiveness analysis. J. Dent. 2015,43, 647–655. [CrossRef]
17.
Mej
à
re, I.; Källest l, C.; Stenlund, H. Incidence and progression of approximal caries from 11 to 22 years of
age in Sweden: A prospective radiographic study. Caries Res. 1999,33, 93–100. [CrossRef]
18.
Alm, A.; Wendt, L.K.; Koch, G.; Birkhed, D. Prevalence of approximal caries in posterior teeth in 15-year-old
Swedish teenagers in relation to their caries experience at 3 years of age. Caries Res.
2007
,41, 392–398.
[CrossRef]
19.
Lussi, A.; Jo
ã
o-Souza, S.H.; Megert, B.; Carvalho, T.S.; Baumann, T. The erosive potential of di↵erent drinks,
foodstu↵s and medicines—a vade mecum. Swiss Dent. J. 2019,129, 479–487.
20.
Nihtila, A.; West, N.; Lussi, A.; Bouchard, P.; Ottolenghi, L.; Senekola, E.; Llodra, J.C.; Viennot, S.; Bourgeois, D.
Oral health behavior and lifestyle factors among overweight and non-overweight young adults in europe:
A cross-sectional questionnaire study. Healthc. Basel Switz. 2016,4, 21. [CrossRef]
21.
Featherstone, J.D.B.; Cha↵ee, B.W. The evidence for caries management by risk assessment (CAMBRA
®
).
Adv. Dent. Res. 2018,29, 9–14. [CrossRef]
22.
Kasmaei, P.; Amin Shokravi, F.; Hidarnia, A.; Hajizadeh, E.; Atrkar-Roushan, Z.; Karimzadeh Shirazi, K.;
Montazeri, A. Brushing behavior among young adolescents: Does perceived severity matter. BMC Public
Health 2014,14, 8. [CrossRef]

Microorganisms 2019,7, 319 20 of 24
23.
Erbe, C.; Klees, V.; Ferrari-Peron, P.; Ccahuana-Vasquez, R.A.; Timm, H.; Grender, J.; Cunningham, P.;
Adam, R.; Farrell, S.; Wehrbein, H. A comparative assessment of plaque removal and toothbrushing
compliance between a manual and an interactive power toothbrush among adolescents: A single-center,
single-blind randomized controlled trial. BMC Oral Health 2018,18, 130. [CrossRef]
24.
De Silva, A.M.; Hegde, S.; Akudo Nwagbara, B.; Calache, H.; Gussy, M.G.; Nasser, M.; Morrice, H.R.;
Riggs, E.; Leong, P.M.; Meyenn, L.K.; et al. Community-based population-level interventions for promoting
child oral health. Cochrane Database Syst. Rev. 2016,9, CD009837.
25.
Huang, Y.K.; Lee, W.F.; Wang, M.J.; Chang, Y.H.S.; Tchaou, W.S.; Chang, W.J.; Lee, S.Y.; Sheu, J.R.; Teng, N.C.
Chair-side quantitative oral-microflora screening for assessing familial correlation of periodontal status and
caries prevalence. PLoS ONE 2014,9, e87100. [CrossRef]
26.
American Academy of Pediatric Dentisty. Guideline on Caries-Risk Assessment and Management for Infants,
Children, and Adolescents. 2010. Available online: http://www.aapd.org/media/Policies_Guidelines/G_
CariesRiskAssessment.pdf (accessed on 15 March 2013).
27.
Clinical A↵airs Committee, American Academy of Pediatric Dentistry Guideline on Adolescent Oral Health
Care. Pediatr. Dent. 2015,37, 49–56.
28.
Pitts, N.B.; Ekstrand, K.R. ICDAS Foundation International Caries Detection and Assessment System (ICDAS)
and its International Caries Classification and Management System (ICCMS)—methods for staging of the
caries process and enabling dentists to manage caries. Community Dent. Oral Epidemiol.
2013
,41, e41–e52.
[CrossRef]
29.
Rosenauer, T.; Wagenschwanz, C.; Kuhn, M.; Kensche, A.; Stiehl, S.; Hannig, C. The bleeding on brushing
index: A novel index in preventive dentistry. Int. Dent. J. 2017,67, 299–307. [CrossRef]
30.
Löe, H. The gingival index, the plaque index and the retention index systems. J. Periodontol.
1967
,38, 610–616.
[CrossRef]
31.
Kralik, P.; Ricchi, M. A basic guide to real time PCR in microbial diagnostics: Definitions, parameters, and
everything. Front. Microbiol. 2017,8, 108. [CrossRef]
32.
Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting linear mixed-e↵ects models using lme4. J. Stat. Softw.
2015,67, 1–48. [CrossRef]
33.
Paolantonio, M.; Festa, F.; di Placido, G.; D’Attilio, M.; Catamo, G.; Piccolomini, R. Site-specific
subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients. Am. J. Orthod.
Dentofac. Orthop. 1999,115, 423–428. [CrossRef]
34.
Ristic, M.; Vlahovic Svabic, M.; Sasic, M.; Zelic, O. Clinical and microbiological e↵ects of fixed orthodontic
appliances on periodontal tissues in adolescents. Orthod. Craniofac. Res. 2007,10, 187–195. [CrossRef]
35.
Pan, S.; Liu, Y.; Zhang, L.; Li, S.; Zhang, Y.; Liu, J.; Wang, C.; Xiao, S. Profiling of subgingival plaque biofilm
microbiota in adolescents after completion of orthodontic therapy. PLoS ONE
2017
,12, e0171550. [CrossRef]
36.
Zaura, E.; Keijser, B.J.F.; Huse, S.M.; Crielaard, W. Defining the healthy “core microbiome” of oral microbial
communities. BMC Microbiol. 2009,9, 259. [CrossRef]
37.
Gomez, A.; Nelson, K.E. The oral microbiome of children: Development, disease, and implications beyond
oral health. Microb. Ecol. 2017,73, 492–503. [CrossRef]
38.
Dewhirst, F.E.; Chen, T.; Izard, J.; Paster, B.J.; Tanner, A.C.R.; Yu, W.H.; Lakshmanan, A.; Wade, W.G. The
human oral microbiome. J. Bacteriol. 2010,192, 5002–5017. [CrossRef]
39. Wade, W.G. The oral microbiome in health and disease. Pharmacol. Res. 2013,69, 137–143. [CrossRef]
40.
Carrouel, F.; Viennot, S.; Santamaria, J.; Veber, P.; Bourgeois, D. Quantitative molecular detection of 19
major pathogens in the interdental biofilm of periodontally healthy young adults. Front. Microbiol.
2016
,7.
[CrossRef]
41.
Iwano, Y.; Sugano, N.; Matsumoto, K.; Nishihara, R.; Iizuka, T.; Yoshinuma, N.I.K. Salivary microbial levels
in relation to periodontal status and caries development. J. Periodontal Res. 2010,45, 165–169. [CrossRef]
42.
Sew
ó
n, L.A.; Parvinen, T.H.; Sinisalo, T.V.; Larmas, M.A.; Alanen, P.J. Dental status of adults with and
without periodontitis. J. Periodontol. 1988,59, 595–598. [CrossRef]
43.
Sioson, P.B.; Furgang, D.; Steinberg, L.M.; Fine, D.H. Proximal caries in juvenile periodontitis patients.
J. Periodontol. 2000,71, 710–716. [CrossRef]
44.
Durand, R.; Roufegarinejad, A.; Chandad, F.; Rompr
é
, P.H.; Voyer, R.; Michalowicz, B.S.; Emami, E. Dental
caries are positively associated with periodontal disease severity. Clin. Oral Investig. 2019, 1–9. [CrossRef]

Microorganisms 2019,7, 319 21 of 24
45.
Cha↵ee, B.W.; Featherstone, J.D.B.; Gansky, S.A.; Cheng, J.; Zhan, L. Caries risk assessment item importance.
JDR Clin. Transl. Res. 2016,1, 131–142. [CrossRef]
46.
Rindal, D.B.; Rush, W.A.; Perrin, N.A.; Maupom
é
, G.; Bader, J.D. Outcomes associated with dentists’ risk
assessment. Community Dent. Oral Epidemiol. 2006,34, 381–386. [CrossRef]
47.
The American Academy of Pediatric Dentistry. Caries-Risk Assessment and Management for Infants,
Children, and Adolescents. 2014. Available online: https://www.aapd.org/media/Policies_Guidelines/BP_
CariesRiskAssessment.pdf (accessed on 5 November 2018).
48. Guo, L.; Shi, W. Salivary biomarkers for caries risk assessment. J. Calif. Dent. Assoc. 2013,41, 107–109.
49.
Krasse, B.; Fure, S. Root surface caries: A problem for periodontally compromised patients. Periodontol. 2000
1994,4, 139–147. [CrossRef]
50.
Gross, E.L.; Beall, C.J.; Kutsch, S.R.; Firestone, N.D.; Leys, E.J.; Gri↵en, A.L. Beyond Streptococcus mutans:
Dental caries onset linked to multiple species by 16S rRNA community analysis. PLoS ONE
2012
,7, e47722.
[CrossRef]
51.
Thenisch, N.L.; Bachmann, L.M.; Imfeld, T.; Leisebach Minder, T.; Steurer, J. Are mutans streptococci detected
in preschool children a reliable predictive factor for dental caries risk? A systematic review. Caries Res.
2006
,
40, 366–374. [CrossRef]
52.
Beighton, D. The complex oral microflora of high-risk individuals and groups and its role in the caries
process. Community Dent. Oral Epidemiol. 2005,33, 248–255. [CrossRef]
53.
Johansson, I.; Witkowska, E.; Kaveh, B.; Lif Holgerson, P.; Tanner, A.C.R. The microbiome in populations
with a low and high prevalence of caries. J. Dent. Res. 2016,95, 80–86. [CrossRef]
54.
Eriksson, L.; Lif Holgerson, P.; Esberg, A.; Johansson, I. Microbial Complexes and Caries in 17-Year-Olds
with and without Streptococcus mutans. J. Dent. Res. 2018,97, 275–282. [CrossRef]
55.
Aas, J.A.; Gri↵en, A.L.; Dardis, S.R.; Lee, A.M.; Olsen, I.; Dewhirst, F.E.; Leys, E.J.; Paster, B.J. Bacteria of
dental caries in primary and permanent teeth in children and young adults. J. Clin. Microbiol.
2008
,46,
1407–1417. [CrossRef]
56.
Gross, E.L.; Leys, E.J.; Gasparovich, S.R.; Firestone, N.D.; Schwartzbaum, J.A.; Janies, D.A.; Asnani, K.;
Gri↵en, A.L. Bacterial 16S sequence analysis of severe caries in young permanent teeth. J. Clin. Microbiol.
2010,48, 4121–4128. [CrossRef]
57.
Tanner, A.C.R.; Mathney, J.M.J.; Kent, R.L.; Chalmers, N.I.; Hughes, C.V.; Loo, C.Y.; Pradhan, N.; Kanasi, E.;
Hwang, J.; Dahlan, M.A.; et al. Cultivable anaerobic microbiota of severe early childhood caries. J. Clin.
Microbiol. 2011,49, 1464–1474. [CrossRef]
58.
Conrads, G.; de Soet, J.J.; Song, L.; Henne, K.; Sztajer, H.; Wagner-Döbler, I.; Zeng, A.P. Comparing the
cariogenic species Streptococcus sobrinus and S. mutans on whole genome level. J. Oral Microbiol.
2014
,6,
26189. [CrossRef]
59.
Drucker, D.B.; Shakespeare, A.P.; Green, R.M. The production of dental plaque and caries by the bacterium
Streptococcus salivarius in gnotobiotic WAG/RIJ rats. Arch. Oral Biol. 1984,29, 437–443. [CrossRef]
60.
Burton, J.P.; Drummond, B.K.; Chilcott, C.N.; Tagg, J.R.; Thomson, W.M.; Hale, J.D.F.; Wescombe, P.A.
Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children:
A randomized double-blind, placebo-controlled trial. J. Med. Microbiol. 2013,62, 875–884. [CrossRef]
61.
Kressirer, C.A.; Smith, D.J.; King, W.F.; Dobeck, J.M.; Starr, J.R.; Tanner, A.C.R. Scardovia wiggsiae and its
potential role as a caries pathogen. J. Oral Biosci. 2017,59, 135–141. [CrossRef]
62.
Tanner, A.C.R.; Sonis, A.L.; Lif Holgerson, P.; Starr, J.R.; Nunez, Y.; Kressirer, C.A.; Paster, B.J.; Johansson, I.
White-spot lesions and gingivitis microbiotas in orthodontic patients. J. Dent. Res.
2012
,91, 853–858.
[CrossRef]
63.
Dzidic, M.; Collado, M.C.; Abrahamsson, T.; Artacho, A.; Stensson, M.; Jenmalm, M.C.; Mira, A. Oral
microbiome development during childhood: An ecological succession influenced by postnatal factors and
associated with tooth decay. ISME J. 2018,12, 2292–2306. [CrossRef]
64.
Ho, M.H.; Lamont, R.J.; Xie, H. Identification of streptococcus cristatus peptides that repress expression of
virulence genes in porphyromonas gingivalis. Sci. Rep. 2017,7.[CrossRef]
65.
Teanpaisan, R.; Thitasomakul, S.; Piwat, S.; Thearmontree, A.; Pithpornchaiyakul, W.; Chankanka, O.
Longitudinal study of the presence of mutans streptococci and lactobacilli in relation to dental caries
development in 3–24 month old Thai children. Int. Dent. J. 2007,57, 445–451. [CrossRef]

Microorganisms 2019,7, 319 22 of 24
66.
Piwat, S.; Teanpaisan, R.; Thitasomakul, S.; Thearmontree, A.; Dahl
é
n, G. Lactobacillus species and genotypes
associated with dental caries in Thai preschool children. Mol. Oral Microbiol. 2010,25, 157–164. [CrossRef]
67.
Hamada, S.; Slade, H.D. Biology, immunology, and cariogenicity of streptococcus mutans. Microbiol. Rev.
1980,44, 331–384.
68.
Corby, P.M.; Lyons-Weiler, J.; Bretz, W.A.; Hart, T.C.; Aas, J.A.; Boumenna, T.; Goss, J.; Corby, A.L.; Junior, H.M.;
Weyant, R.J.; et al. Microbial risk indicators of early childhood caries. J. Clin. Microbiol.
2005
,43, 5753–5759.
[CrossRef]
69.
Caufield, P.W.; Schön, C.N.; Saraithong, P.; Li, Y.; Argim
ó
n, S. Oral lactobacilli and dental caries: A model for
niche adaptation in humans. J. Dent. Res. 2015,94, 110S–118S. [CrossRef]
70.
Modesto, M.; Biavati, B.; Mattarelli, P. Occurrence of the family bifidobacteriaceae in human dental caries
and plaque. Caries Res. 2006,40, 271–276. [CrossRef]
71.
Tanner, A.C.R.; Kent, R.L.; Holgerson, P.L.; Hughes, C.V.; Loo, C.Y.; Kanasi, E.; Chalmers, N.I.; Johansson, I.
Microbiota of severe early childhood caries before and after therapy. J. Dent. Res.
2011
,90, 1298–1305.
[CrossRef]
72.
Nair, S.; Kumar, V.S.; Krishnan, R.; Rajan, P. A comparative evaluation of bifidobacteria levels in early
childhood caries and severe early childhood caries. J. Pharm. Bioallied Sci. 2017,9, S82–S84. [CrossRef]
73.
Manome, A.; Abiko, Y.; Kawashima, J.; Washio, J.; Fukumoto, S.; Takahashi, N. Acidogenic potential of oral
bifidobacterium and its high fluoride tolerance. Front. Microbiol. 2019,10, 1099. [CrossRef]
74.
Jiang, S.; Gao, X.; Jin, L.; Lo, E.C.M. Salivary microbiome diversity in caries-free and caries-a↵ected children.
Int. J. Mol. Sci. 2016,17, 1978. [CrossRef]
75.
Ferrazzano, G.F.; Scioscia, E.; Sateriale, D.; Pastore, G.; Colicchio, R.; Pagliuca, C.; Cantile, T.; Alcidi, B.;
Coda, M.; Ingenito, A.; et al.
In vitro
antibacterial activity of pomegranate juice and peel extracts on cariogenic
bacteria. BioMed Res. Int. 2017,2017, 2152749. [CrossRef]
76.
Hajishengallis, E.; Parsaei, Y.; Klein, M.I.; Koo, H. Advances in the microbial etiology and pathogenesis of
early childhood caries. Mol. Oral Microbiol. 2017,32, 24–34. [CrossRef]
77.
Xu, H.; Tian, J.; Hao, W.; Zhang, Q.; Zhou, Q.; Shi, W.; Qin, M.; He, X.; Chen, F. Oral microbiome shifts from
caries-free to caries-a↵ected status in 3-year-old chinese children: A longitudinal study. Front. Microbiol.
2018,9, 2009. [CrossRef]
78.
Hajishengallis, G. Immunomicrobial pathogenesis of periodontitis: Keystones, pathobionts, and host
response. Trends Immunol. 2014,35, 3–11. [CrossRef]
79.
Tu, Y.; Ling, X.; Chen, Y.; Wang, Y.; Zhou, N.; Chen, H. E↵ect of S. Mutans and S. Sanguinis on growth and
adhesion of P. Gingivalis and their ability to adhere to di↵erent dental materials. Med. Sci. Monit. Int. Med. J.
Exp. Clin. Res. 2017,23, 4539–5445. [CrossRef]
80.
Hua, X.; Guy, S.; Cook, J.; William, C.; Greg, B.; Timothy, M.; Richard, J.; Lamont, R.J. Intergeneric
communication in dental plaque biofilms. J. Bacteriol. 2000,128, 7067–7069.
81.
Zhu, Y.; Dashper, S.G.; Chen, Y.Y.; Crawford, S.; Slakeski, N.; Reynolds, E.C. Porphyromonas gingivalis and
Treponema denticola synergistic polymicrobial biofilm development. PLoS ONE
2013
,8, e71727. [CrossRef]
82.
Cogoni, V.; Morgan-Smith, A.; Fenno, J.C.; Jenkinson, H.F.; Dymock, D. Treponema denticola chymotrypsin-
like proteinase (CTLP) integrates spirochaetes within oral microbial communities. Microbiol. Read. Engl.
2012,158, 759–770. [CrossRef]
83.
Peterson, S.N.; Snesrud, E.; Liu, J.; Ong, A.C.; Kilian, M.; Schork, N.J.; Bretz, W. The dental plaque microbiome
in health and disease. PLoS ONE 2013,8, e58487. [CrossRef]
84.
Sharma, A.; Inagaki, S.; Sigurdson, W.; Kuramitsu, H.K. Synergy between Tannerella forsythia and
Fusobacterium nucleatum in biofilm formation. Oral Microbiol. Immunol. 2005,20, 39–42. [CrossRef]
85.
Nath, S.G.; Raveendran, R. Microbial dysbiosis in periodontitis. J. Indian Soc. Periodontol.
2013
,17, 543–545.
[CrossRef]
86.
Kanasi, E.; Johansson, I.; Lu, S.C.; Kressin, N.R.; Nunn, M.E.; Kent, R.; Tanner, A.C.R. Microbial risk markers
for childhood caries in pediatricians’ offices. J. Dent. Res. 2010,89, 378–383. [CrossRef]
87.
Nakagawa, S.; Fujii, H.; Machida, Y.; Okuda, K. A longitudinal study from prepuberty to puberty of gingivitis.
Correlation between the occurrence of Prevotella intermedia and sex hormones. J. Clin. Periodontol.
1994
,21,
658–665. [CrossRef]
88.
Lee, H.J.; Kim, J.K.; Cho, J.Y.; Lee, J.M.; Hong, S.H. Quantification of subgingival bacterial pathogens at
di↵erent stages of periodontal diseases. Curr. Microbiol. 2012,65, 22–27. [CrossRef]

Microorganisms 2019,7, 319 23 of 24
89.
Becker, M.R.; Paster, B.J.; Leys, E.J.; Moeschberger, M.L.; Kenyon, S.G.; Galvin, J.L.; Boches, S.K.; Dewhirst, F.E.;
Gri↵en, A.L. Molecular analysis of bacterial species associated with childhood caries. J. Clin. Microbiol.
2002
,
40, 1001–1009. [CrossRef]
90.
Belda-Ferre, P.; Alcaraz, L.D.; Cabrera-Rubio, R.; Romero, H.; Sim
ó
n-Soro, A.; Pignatelli, M.; Mira, A. The
oral metagenome in health and disease. ISME J. 2012,6, 46–56. [CrossRef]
91.
Bik, E.M.; Long, C.D.; Armitage, G.C.; Loomer, P.; Emerson, J.; Mongodin, E.F.; Nelson, K.E.; Gill, S.R.;
Fraser-Liggett, C.M.; Relman, D.A. Bacterial diversity in the oral cavity of 10 healthy individuals. ISME J.
2010,4, 962–974. [CrossRef]
92.
Aas, J.A.; Paster, B.J.; Stokes, L.N.; Olsen, I.; Dewhirst, F.E. Defining the normal bacterial flora of the oral
cavity. J. Clin. Microbiol. 2005,43, 5721–5732. [CrossRef]
93.
Abusleme, L.; Dupuy, A.K.; Dutzan, N.; Silva, N.; Burleson, J.A.; Strausbaugh, L.D.; Gamonal, J.; Diaz, P.I.
The subgingival microbiome in health and periodontitis and its relationship with community biomass and
inflammation. ISME J. 2013,7, 1016–1025. [CrossRef]
94.
Ge, Y.; Caufield, P.W.; Fisch, G.S.; Li, Y. Streptococcus mutans and Streptococcus sanguinis colonization
correlated with caries experience in children. Caries Res. 2008,42, 444–448. [CrossRef]
95.
Choi, H.; Kim, E.; Kang, J.; Kim, H.J.; Lee, J.Y.; Choi, J.; Joo, J.Y. Real-time PCR quantification of 9 periodontal
pathogens in saliva samples from periodontally healthy Korean young adults. J. Periodontal Implant Sci.
2018
,
48, 261–271. [CrossRef]
96.
Suda, R.; Lai, C.H.; Yang, H.W.; Hasegawa, K. Eikenella corrodens in subgingival plaque: Relationship to
age and periodontal condition. J. Periodontol. 2002,73, 886–891. [CrossRef]
97.
Noiri, Y.; Li, L.; Ebisu, S. The localization of periodontal-disease-associated bacteria in human periodontal
pockets. J. Dent. Res. 2001,80, 1930–1934. [CrossRef]
98.
Dye, B.A.; Tan, S.; Smith, V.; Lewis, B.G.; Barker, L.K.; Thornton-Evans, G.; Eke, P.I.; Beltr
á
n-Aguilar, E.D.;
Horowitz, A.M.; Li, C.H. Trends in oral health status: United States, 1988–1994 and 1999–2004. Vital Health
Stat. 11 2007, 1–92.
99.
Yin, W.; Yang, Y.M.; Chen, H.; Li, X.; Wang, Z.; Cheng, L.; Yin, Q.D.; Fang, H.Z.; Fei, W.; Mi, F.L.; et al. Oral
health status in Sichuan Province: Findings from the oral health survey of Sichuan, 2015–2016. Int. J. Oral
Sci. 2017,9, 10–15. [CrossRef]
100.
Hou, R.; Mi, Y.; Xu, Q.; Wu, F.; Ma, Y.; Xue, P.; Xiao, G.; Zhang, Y.; Wei, Y.; Yang, W. Oral health survey and
oral health questionnaire for high school students in Tibet, China. Head Face Med. 2014,10, 17. [CrossRef]
101.
Hu, J.; Jiang, W.; Lin, X.; Zhu, H.; Zhou, N.; Chen, Y.; Wu, W.; Zhang, D.; Chen, H. Dental caries status and
caries risk factors in students ages 12–14 years in Zhejiang, China. Med. Sci. Monit. Int. Med. J. Exp. Clin. Res.
2018,24, 3670–3678. [CrossRef]
102.
Yetimoglu, C.; Rafeiner, P.; Engel, D.; Fournier, J.Y. Spinal infections due to Eikenella corrodens: Case report
and literature review. Neurochirurgie. 2014,60, 197–200. [CrossRef]
103.
Udaka, T.; Hiraki, N.; Shiomori, T.; Miyamoto, H.; Fujimura, T.; Inaba, T.; Suzuki, H. Eikenella corrodens in
head and neck infections. J. Infect. 2007,54, 343–348. [CrossRef]
104.
Angulo L
ó
pez, I.; Aguirre Quiñonero, A.; Fern
á
ndez Torres, M.; Alegr
í
a Echauri, E. Chorioamnionitis and
neonatal septicaemia due to Eikenella corrodens. Enferm. Infecc. Microbiol. Clin.
2017
,35, 266–267. [CrossRef]
105.
Sheng, W.S.; Hsueh, P.R.; Hung, C.C.; Teng, L.J.; Chen, Y.C.; Luh, K.T. Clinical features of patients with
invasive Eikenella corrodens infections and microbiological characteristics of the causative isolates. Eur. J.
Clin. Microbiol. Infect. Dis. 2001,20, 231–236. [CrossRef]
106.
Elikowski, W.; Małek-Elikowska, M.; Lisiceka, M.; Wr
ó
blewski, D.; Fertała, N. Eikenella corrodens
endocarditis of the tricuspid valve in an intravenous drug user. Pol. Merkur. Lek. Organ Pol. Tow. Lek.
2017
,
42, 81–83.
107.
Kanasi, E.; Dewhirst, F.E.; Chalmers, N.I.; Kent, R.; Moore, A.; Hughes, C.V.; Pradhan, N.; Loo, C.Y.;
Tanner, A.C.R. Clonal analysis of the microbiota of severe early childhood caries. Caries Res.
2010
,44, 485–497.
[CrossRef]
108.
Luppens, S.B.I.; Kara, D.; Bandounas, L.; Jonker, M.J.; Wittink, F.R.A.; Bruning, O.; Breit, T.M.; Ten Cate, J.M.;
Crielaard, W. E↵ect of veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus
mutans grown in a dual-species biofilm. Oral Microbiol. Immunol. 2008,23, 183–189. [CrossRef]

Microorganisms 2019,7, 319 24 of 24
109.
Liljemark, W.F.; Bloomquist, C.G.; Bandt, C.L.; Pihlstrom, B.L.; Hinrichs, J.E.; Wol↵, L.F. Comparison of the
distribution of actinomyces in dental plaque on inserted enamel and natural tooth surfaces in periodontal
health and disease. Oral Microbiol. Immunol. 1993,8, 5–15. [CrossRef]
110.
Tanner, A.C.R.; Milgrom, P.M.; Kent, R.; Mokeem, S.A.; Page, R.C.; Riedy, C.A.; Weinstein, P.; Bruss, J. The
microbiota of young children from tooth and tongue samples. J. Dent. Res. 2002,81, 53–57. [CrossRef]
111.
Marchant, S.; Brailsford, S.R.; Twomey, A.C.; Roberts, G.J.; Beighton, D. The predominant microflora of
nursing caries lesions. Caries Res. 2001,35, 397–406. [CrossRef]
112.
Li, Y.; Zou, C.G.; Fu, Y.; Li, Y.; Zhou, Q.; Liu, B.; Zhang, Z.; Liu, J. Oral microbial community typing of caries
and pigment in primary dentition. BMC Genomics 2016,17, 558. [CrossRef]
113.
Sampaio-Maia, B.; Caldas, I.M.; Pereira, M.L.; P
é
rez-Mongiovi, D.; Araujo, R. The Oral microbiome in health
and its implication in oral and systemic diseases. Adv. Appl. Microbiol. 2016,97, 171–210.
114.
Jia, G.; Zhi, A.; Lai, P.F.H.; Wang, G.; Xia, Y.; Xiong, Z.; Zhang, H.; Che, N.; Ai, L. The oral microbiota—A
mechanistic role for systemic diseases. Br. Dent. J. 2018,224, 447–455. [CrossRef]
115.
Weber, E.; Reynaud, Q.; Suy, F.; Gagneux-Brunon, A.; Carricajo, A.; Guillot, A.; Botelho-Nevers, E.
Bifidobacterium species bacteremia: Risk factors in adults and infants. Clin. Infect. Dis. O↵. Publ.
Infect. Dis. Soc. Am. 2015,61, 482–484. [CrossRef]
116.
Könönen, E.; Wade, W.G. Actinomyces and related organisms in human infections. Clin. Microbiol. Rev.
2015
,
28, 419–442. [CrossRef]
117.
Datta, S.; Soliman, M.; Laguio, M. Actinomyces in blood: Is it clinically significant or insignificant? Open Forum
Infect. Dis. 2017,4, S554. [CrossRef]
118.
Binder, D.; Zbinden, R.; Widmer, U.; Opravil, M.; Krause, M. Native and prosthetic valve endocarditis caused
by Rothia dentocariosa: Diagnostic and therapeutic considerations. Infection 1997,25, 22–26. [CrossRef]
119.
Dhotre, S.V.; Davane, M.S.; Nagoba, B.S. Periodontitis, bacteremia and infective endocarditis: A review study.
Arch. Pediatr. Infect. Dis. 2017,5, e41067. [CrossRef]
120.
Gondivkar, S.; Gadbail, A.; Parikh, R.V. Role of oral foci in systemic diseases: An update. Int. J. Contemp.
Dent. Med. Rev. 2017, 1–8.
121.
Nordholm, A.C.; Vøgg, R.O.B.; Permin, H.; Katzenstein, T. Eikenella corrodens endocarditis and liver abscess
in a previously healthy male, a case report. BMC Infect. Dis. 2018,18, 35. [CrossRef]
122.
Beck, J.D.; Eke, P.; Heiss, G.; Madianos, P.; Couper, D.; Lin, D.; Moss, K.; Elter, J.; O↵enbacher, S. Periodontal
disease and coronary heart disease: A reappraisal of the exposure. Circulation 2005,112, 19–24. [CrossRef]
123.
Bagavant, H.; Dunkleberger, M.L.; Wolska, N.; Sroka, M.; Rasmussen, A.; Adrianto, I.; Montgomery, C.;
Sivils, K.; Guthridge, J.M.; James, J.A.; et al. Antibodies to periodontogenic bacteria are associated with
higher disease activity in lupus patients. Clin. Exp. Rheumatol. 2019,37, 106–111.
124.
Bui, F.Q.; Almeida-da-Silva, C.L.C.; Huynh, B.; Trinh, A.; Liu, J.; Woodward, J.; Asadi, H.; Ojcius, D.M.
Association between periodontal pathogens and systemic disease. Biomed. J. 2019,42, 27–35. [CrossRef]
125.
Larsen, J.M. The immune response to Prevotella bacteria in chronic inflammatory disease. Immunology
2017
,
151, 363–374. [CrossRef]
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