Application of Denaturing Gradient Gel

Electrophoresis to the Analysis of

Bacterial Communities Associated With
Asymptomatic and Symptomatic
Pericoronitis
Xin Zhang, MD,* Zheng Sun, PhD,y and Qiubo Yang, PhDz
Purpose: Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial commu-
nities associated with asymptomatic and symptomatic pericoronitis. The aim of the study was to compare
the fingerprinting patterns of these 2 clinical conditions.
Materials and Methods: The microbiota of mandibular third molar pockets associated with asymptom-
atic or symptomatic pericoronitis cases were collected and profiled by the polymerase chain reaction
DGGE method. Banding patterns were compared by cluster analysis techniques.
Results: Thirteen symptomatic pericoronitis and 7 asymptomatic pericoronitis samples were collected.
Comparative analysis of the 2 clinical conditions showed bands that were common to the symptomatic and
asymptomatic cases, but most DGGE bands appeared to be unique to the clinical condition. No single band
occurred in all profiles. The mean number of bands detected in the 16S rDNA community profiles was
23.8  4.2 (range, 19 to 34) for samples from symptomatic cases and 24.1  2.4 (range, 21 to 29) for those
from asymptomatic cases. Cluster analysis and multidimensional scaling analysis of the DGGE banding pattern
showed a distinction in the similarity of banding patterns according to the presence or absence of symptoms.
Conclusions: These results suggest that the diversity of pericoronal pocket microbiota in asymptomatic
pericoronitis cases differs markedly from that of symptomatic cases.
Ó 2017 Published by Elsevier Inc on behalf of the American Association of Oral and Maxillofacial
Surgeons
J Oral Maxillofac Surg 76:483-489, 2018

Pericoronitis is characterized by inflammation around similar to those found in gingivitis or periodonti-

the partly erupted mandibular third molar and is tis cases.3
ranked as the leading cause1 of acute oral health prob- A comprehensive analysis of the microbial diver-
lems in young adults. The exposure of partly erupted sity in different environments requires methods
teeth to the oral cavity environment can result in that do not depend on cultivation. A great deal of sci-
pathology.2 The microbes and mainly anaerobic entific evidence has shown that most micro-
microflora found associated with pericoronitis are organisms in nature are not cultivable because of

Received from the Beijing Stomatological Hospital and School of and School of Stomatology, Capital Medical University, Tiantan Xili
Stomatology, Capital Medical University, Beijing, China. 4#, Dongcheng District, Beijing 100050, People’s Republic of China;
*Doctor, Department of Dental Emergency. e-mail: [email protected]
yProfessor, Department of Oral Medicine. Received March 8 2017
zProfessor, Beijing Institute for Dental Research. Accepted August 5 2017
This work was supported by the National Natural Science Foun- Ó 2017 Published by Elsevier Inc on behalf of the American Association of Oral
dation of China (grants 30572037 and 81470753) and the Beijing and Maxillofacial Surgeons
Natural Science Foundation (grants 7062028 and 7122078). 0278-2391/17/31066-2
Conflict of Interest Disclosures: None of the authors have a rele- http://dx.doi.org/10.1016/j.joms.2017.08.007
vant financial relationship(s) with a commercial interest.
Address correspondence and reprint requests to Prof Yang: Bei-
jing Institute for Dental Research, Beijing Stomatological Hospital

483
484 ANALYSIS OF BACTERIAL COMMUNITIES USING DGGE

the attempt to separate bacteria in solid culture me- Materials and Methods
dium, which is likely to disrupt intercommunica-
PATIENTS
tions.4 The development of molecular technologies
has allowed the identification of micro-organisms This study was conducted in accordance with the
and the study of microbial diversity at the genetic Declaration of Helsinki and with approval from the
level. Genetic fingerprinting techniques can be ethics committee of Capital Medical University (Bei-
used as alternatives to cloning, because they provide jing, China). Written informed consent was obtained
a profile of the genetic diversity of a microbial com- from all participants. The study population was
munity in a given environment. Denaturing gradient composed of 13 patients presenting to the Emergency
gel electrophoresis (DGGE) has been one of the Department at Beijing Stomatological Hospital (Bei-
most commonly used techniques to fingerprint mi- jing, China) for the evaluation and management of
crobial communities.5 DGGE is an electrophoretic pericoronitis (7 men and 6 women; age range, 18 to
method that can detect differences between DNA 30 years). To be included in the study, patients had
fragments of the same size but with different se- to be examined clinically and with radiography;
quences and that has great potential for the identifi- healthy periodontium surrounding teeth other than
cation of closely related species based on 16S rDNA the third molars was the principal inclusion criterion
sequences. Furthermore, separation of polymerase for the study. Patients were excluded as study subjects
chain reaction (PCR) products by DGGE results in if antimicrobial therapy was conducted within the
a banding pattern, with the number of bands usually past 30 days.
corresponding to the number of predominant mem-
bers in the microbial community.6 Statistical STUDY SAMPLES
methods, such as clustering techniques and prin- Samples were obtained from patients with the
cipal component analyses, can be applied to DGGE following clinical diagnoses: symptomatic pericoroni-
profiles with the aim of identifying samples that tis with acute pericoronitis and exhibiting localized
generate similar banding patterns. One advantage or diffuse swellings in addition to fever, lymphadenop-
of this presentation is that similarities in finger- athy, or malaise (P group; n = 13); and asymptomatic
printing patterns can be assessed rapidly.7 These pericoronitis with chronic pericoronitis and not
methods have often been used for the interpretation showing localized or diffuse swellings in addition to fe-
of DGGE community fingerprinting profiles.8-10 ver, lymphadenopathy, or malaise (ASY group; n = 7).
As a molecular tool, DGGE is reliable, reproducible,
rapid, and inexpensive11 and allows the study of the SPECIMEN COLLECTION
complexity and behavior of microbial communities
Contamination was carefully prevented by cotton
over time. DGGE has been recently applied to finger-
swabs. A sterile endodontic paper point was inserted
printing microbial communities associated with hu-
into the depth of the pericoronal pocket and left in
man clinical samples, including those occurring in
place for 30 seconds. Three paper points were used
subgingival plaque from healthy patients or those
for each pocket. Then, the paper points were inserted
with marginal periodontitis,12,13 microbiota
into an Eppendorf tube containing sterile saline 1 mL
associated with acute apical abscesses from Brazilian
and the tubes were stored at 20 C.
and US patients,14 and pooled dental plaque samples
from children with severe early childhood caries and
DNA EXTRACTION
children without caries.15 One of the greatest advan-
tages of the DGGE method is that the fingerprint gives DNA was extracted from the archived periodontal
a ‘‘picture’’ of the microbiota present in a given sam- samples with a DNeasy Tissue Kit: Wizard Genomic
ple.4,5 Although DGGE has been applied to analyze DNA Purification Kit (Promega, Madison, WI) in accor-
microbial communities from a plethora of dance with the manufacturer’s instructions.
environments, this technique has not yet been used
to study the microbiology of pericoronitis. NESTED PCR
The purpose of this study was to investigate the bac- Nested PCR was performed with 2 sets of universal
terial community profile of microbiota in asymptom- primers. The complete 1,500-bp 16S rRNA gene locus
atic and symptomatic pericoronitis using DGGE. The was amplified from DNA extracts of all pericoronitis
authors hypothesized there would be differences in samples with a set of universal 16S rRNA gene
the diversity of overall bacterial community profiles sequence primers: 27F (50 -AGA GTT TGA TCC TGG
between asymptomatic and symptomatic pericoroni- CTC AG-30 ) and 1492R (50 -GGT TAC CTT GTT ACG
tis. The specific aims of the study were to compare ACT T-30 ; AuGCT, Beijing, China). In the nested round
the DGGE fingerprinting patterns of these 2 clinical of the PCR, a second set of universal bacterial 16S
conditions. rRNA gene primers (HDA1 and HDA2) was used with
ZHANG, SUN, AND YANG 485

a 40-nucleotide GC clamp that was added to the 50 end DENDROGRAM CONSTRUCTION AND CLUSTER
of HDA1 to prevent the dissociation of the 16S rRNA ANALYSIS
gene duplexes during denaturation electrophoresis. Gel images were analyzed using BioNumerics soft-
The V2 to V3 region of the 16S rRNA gene (corre- ware (Applied Maths, Austin, TX). A minimal
sponding to positions 339 to 539 of the Escherichia profiling setting (3.0%) was used for band searching
coli genome) was amplified with Eubacterium-spe- in all DGGE gels. DGGE profiles were determined
cific primers HDA1-GC (50 -CGC CCG GGG CGC GCC by the number of detected bands, the migration dis-
CCG GGC GGG GCG GGG GCA CGG GGG GAC tances, and the intensities of the bands within each
TCC TAC GGG AGG CAG CAG T-30 ) and HDA2 (50 - lane. Then, each lane on the gel was compared
GTA TTA CCG CGG CTG CTG GCA C-30 ) as previously with the reference lane, allowing a matching profile
described.16 for each lane to be generated. Band search filters
The PCR mixture was composed of DNA 4 mL ex- were used to define bands as minimum profiling,
tracted from the clinical samples, universal primer ‘‘gray zone,’’ shoulder sensitivity, and minimum area.
stock 4 mL, 2 Hotstart Taq PCR MasterMix 25 mL A pairwise similarity index (Cs) was calculated for
(Tiangen, Beijing, China), and sterile filtered Milli-Q each sample. The percentage of similarity between
water for a final volume of 50 mL. All PCR amplifica- fingerprinting profiles was calculated according to
tions were performed in an Eppendorf thermocycler the Dice coefficient of pairwise comparisons. The
(MWG-Biotech, Ebersberg, Germany). The reaction Ward algorithm was used to construct a dendrogram
conditions included an initial denaturation step at for cluster analysis.17
95 C (5 minutes), followed by 35 cycles of a denatur- Cluster analysis was conducted to determine
ation step at 94 C (60 seconds), a primer annealing whether the samples presented a nonrandom pattern
step at 56 C (60 seconds), and an extension step at and whether they clustered according to the presence
72 C (30 seconds). The final cycle incorporated an 8- or absence of symptoms. Multidimensional scaling
minute chain elongation step (72 C). Before DGGE (MDS) was used to investigate the variation in DGGE
analysis, the presence of PCR products was confirmed banding patterns. The high-dimensional relations be-
by electrophoresis in a 1% agarose gel conducted at tween samples were represented in a 3-dimensional
120 V in 5 TBE (Sangon Biotech, Shanghai, China) plot. In this plot, similar samples were placed close
buffer for 30 minutes. The gel was stained for 15 mi- together, and the distance between samples increased
nutes with Goldview Nucleic Acid Stain (SBS Gene- as genetic similarity decreased.18
tech, Beijing, China) and viewed with GeneGenius
(Gene Snap software, Syngene, UK) under 300-nm ul-
STATISTICAL ANALYSES
traviolet light. DNA Marker I (Tiangen) served as the
molecular size standard. Differences in microbial diversity were assessed by
comparing the DGGE profiles within and between
the P and ASY groups. The correlation between the
DGGE ASSAY microbial diversity and the clinical condition of the
The final PCR products derived from community patients was evaluated with analysis of variance (AN-
samples were resolved with a Bio-Rad DCode Univer- OVA), nonparametric Mann-Whitney U test, and
sal Mutation Detection System (Bio-Rad, Hercules, Fisher exact test. Analyses were performed with
CA). The PCR products (30 mL) were loaded onto 8% SPSS Statistics 17.0 (SPSS, Inc, Chicago, IL). A 2-
(w/v) polyacrylamide gels (acrylamide-to-bis ratio, tailed test with a P value less than .05 was considered
37.5:1; 16  16 cm, 1 mm deep) containing a linear significant.
gradient of denaturant ranging from 30 to 60%, with
100% denaturant corresponding to urea 7.0 mol/L Results
and 40% (v/v) formamide, increasing in the direction
of electrophoresis. Denaturing gradients were formed GENERAL INFORMATION
with Gradient Former (Bio-Rad). The direction of inclined third molars included
The electrophoresis ran in 1 TAE buffer diluted from distal, vertical, and mesial. Pericoronitis most likely
50 TAE buffer (Tris base 40 mmol/L, glacial acetic acid occurs when there is vertical or distal inclination of
20 mmol/L, ethylenediaminetetra-acetic acid 1 mmol/L) the mandibular third molar. The degree of eruption
at 60 V and 60 C for 15 hours. All gels were stained was defined as higher or lower than the occlusal sur-
with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, face of the adjacent second molar. The visible
CA) for 30 minutes. Gels were viewed with Quantity occlusal surface of the partly erupted teeth was
One Gel Doc XR (Bio-Rad) and images were digitally defined as more or less than 50% of the occlusal sur-
captured with Quantity One 4.6.3 software (Bio-Rad). face. The direction of inclined third molars and the
Three lanes of the same sample were run in a single gel. degree of eruption were not statistically different
486 ANALYSIS OF BACTERIAL COMMUNITIES USING DGGE

Table 1. COMPARISONS OF THE DIVERSITY OF DGGE PROFILES IN ASYMPTOMATIC AND SYMPTOMATIC

PERICORONITIS

Outcome Variable P Group (n = 13) ASY Group (n = 7) P Value

Clinical data, n (%)

Distal/vertical/mesial* 10/1/2 (77/8/15) 5/0/2 (71/0/29) 1.000x
Higher/lowery 8/5 (62/38) 5/2 (71/29) 1.000x
More/lessz 3/10 (23/77) 6/1 (86/14) .017x
Microbial diversity, mean  SD
DGGE bands (n) 23.8  4.2 24.1  2.4 .577k
Similarity values (%), mean  SD
Intergroup comparison 57.6  8.3 64.0  8.4 .162{
Intragroup comparison 58.9  7.6 .006{

Abbreviations: ASY, asymptomatic pericoronitis; DGGE, denaturing gradient gel electrophoresis; P, symptomatic pericoronitis;
SD, standard deviation.
* Distally, vertically, or mesially inclined third molars.
y Level of eruption higher or lower than the occlusal surface of the adjacent second molar.
z Visible occlusal surface of the partly erupted teeth more or less than 50% of the occlusal surface.
x By Fisher exact test.
k By nonparametric Mann-Whitney U test for independent samples.
{ Results of analysis of variance showed that the P value for the overall comparison was .006. Significant differences also were
found between the P group and the intragroup comparison (57.6 vs 58.9; P = .319) and between the ASY group and the intra-
group comparison (64.0 vs 58.9; P = .008).
Zhang, Sun, and Yang. Analysis of Bacterial Communities Using DGGE. J Oral Maxillofac Surg 2018.

between the 2 groups as determined by the Fisher CLUSTER ANALYSIS

exact test. In contrast, the visible occlusal surface be- Figure 2 depicts the results of the Ward analysis in
tween the 2 groups was significantly different which the Dice coefficient for measuring similarity
(P = .017; Table 1). in banding patterns was applied. The ASY and P groups
displayed a statistically significant clustering of pro-
files, resolving into cluster 1 (P group) and cluster 2
DGGE BANDING PATTERN
(ASY group; P = .003 by Fisher exact test).
DGGE profiles were obtained from samples of the
20 patients (P group, n = 13; ASY group, n = 7; MDS ANALYSIS
Fig 1). Sixty-nine distinct amplicons (bands) were de-
Cluster analysis of DGGE band polymorphisms
tected in the DGGE profiles after gel normalization.
clearly separated ASY from P cases. Except for 1
The number of distinct amplicons ranged from 19 to
sample, samples from ASY cases tended to cluster
34 (mean  standard deviation, 27.3  3.6) for each
with their respective type. Samples from P cases uni-
sample. The mean number of bands in symptomatic
formly clustered together. These findings were
cases was smaller than in asymptomatic cases. In the
confirmed by MDS with 3-dimensional scaling
16S rDNA community profiles, there were
(Fig 3).
23.8  4.2 (range, 19 to 34) and 24.1  2.4 (range,
21 to 29) bands in samples from the P and ASY cases,
respectively. However, this difference was not signifi-
Discussion
cant (P = .577 by Mann-Whitney U test; Table 1). Intra-
group and intergroup comparisons of the microbial This study investigated the bacterial community
profiles showed that the mean similarity values were profile of microbiota in asymptomatic and symptom-
57.6  8.3% within the P group samples and atic pericoronitis using DGGE. The authors hypothe-
64.0  8.4% within the ASY group samples, and that sized there would be differences in diversity of
the similarity values of the 2 groups was significantly overall bacterial community profiles between asymp-
different (58.9  7.6%; P = .006 by ANOVA; Table 1). tomatic and symptomatic pericoronitis. They
Distinct banding patterns were observed in profiles compared the DGGE fingerprinting patterns of 2 clin-
from the various clinical samples (Fig 1). Most profiles ical conditions and found that pericoronitis is a poly-
contained intense DNA bands and many faint DNA microbial disease and there were notable differences
bands. Some profiles consisted almost exclusively of in the bacterial community composition between
faint bands. No single band occurred in all profiles. ASY and P cases.
ZHANG, SUN, AND YANG 487

FIGURE 1. Denaturing gradient gel electrophoresis profiles of bacterial 16S rRNA gene segments amplified by polymerase chain reaction.
Denaturing gradient gel electrophoresis images were obtained from the total genomic DNAs of samples from the pockets of 13 patients with
symptomatic pericoronitis and 7 with asymptomatic pericoronitis. M, marker.
Zhang, Sun, and Yang. Analysis of Bacterial Communities Using DGGE. J Oral Maxillofac Surg 2018.

Rajasuo et al19 reported that in patients with healthy Recent developments in molecular analytical
periodontal tissue third molar follicles harbor the same methods have allowed researchers to overcome
bacterial species that are common in periodontitis. many of the restrictions associated with culture-
Pericoronitis is a polymicrobial infectious disease; based techniques when studying microbial ecosystems
however, further studies are needed to determine associated with the periodontal pocket. The bacterial
whether microbial communities from pericoronitis- diversity in healthy and diseased human subgingival
associated plaques are more or less diverse than pla- crevices has been investigated by combining DGGE
ques that are not associated with pericoronitis and of the 16S rRNA gene with image and cluster analyses
to elucidate the determinants associated with these and sequencing of genome regions, statistical analyses,
community compositions. and multiplex PCR of 3 periodontal pathogens.17

FIGURE 2. Cluster analysis. The difference in microbial diversity was clearly distinguished by cluster analysis with the Ward algorithm based
on the Dice coefficient. A distinct cluster (cluster 1) from the P group was observed, and 11 of 13 P profiles were grouped into 1 dendrogram
branch (P = .003 by Fisher exact test). Denaturing gradient gel electrophoresis profiles of the ASY group were differentiated from those of the P
group in a separate cluster. The difference in mean similarity values (58.9  7.6%; P = .006 by analysis of variance) between the 2 groups was
statistically significant. ASY, asymptomatic pericoronitis; P, symptomatic pericoronitis.
Zhang, Sun, and Yang. Analysis of Bacterial Communities Using DGGE. J Oral Maxillofac Surg 2018.
488 ANALYSIS OF BACTERIAL COMMUNITIES USING DGGE

delineation of these 2 clusters reflected distinctions

in the number of bands detected (richness), the inten-
sity of bands, and the migration distribution of the am-
plicons. As expected, there was considerably greater
phylogenetic relatedness within each group than be-
tween the 2 groups. This suggests that different
ecologic factors contributed to shaping the commu-
nity within individuals.23 This finding is in line with
the DGGE profiling results from similarly obtained
samples,24 in which high inter- and intraindividual var-
iabilities among sample profiles were observed.
In this study, the banding profiles in the DGGE gels
were analyzed by MDS. The highest dispersion of the
samples is represented in the 3-dimensional plot.
MDS applied to the presence or absence of bands
within DGGE patterns confirmed that the composition
of the pericoronitis microbiota differed between
symptomatic and asymptomatic cases.
In the present study, the DGGE method was applied
to examine the structure of bacterial communities in
FIGURE 3. Multidimensional scaling analysis score plots of dena-
turing gradient gel electrophoresis banding patterns from samples samples taken from ASY and P cases. DNA banding pat-
taken from pockets associated with asymptomatic or symptomatic terns from any case showed relative heterogeneity to
pericoronitis. Two distinct clusters (open circles, cluster 1; solid others. Admittedly, the number of DGGE bands in a
squares, cluster 2) were observed. A, asymptomatic pericoronitis;
P, symptomatic pericoronitis. pattern is related to the number of bacterial species
Zhang, Sun, and Yang. Analysis of Bacterial Communities Using
in the consortium.6 Sample profiles were not identical,
DGGE. J Oral Maxillofac Surg 2018. even within the ASY or P groups. One assumption in
the interpretation of DGGE fingerprinting is that the
Siqueira et al20 investigated the bacterial communities band intensity is directly related to the density of cor-
associated with asymptomatic and symptomatic end- responding bacterial phylotypes within the sample.25
odontic infections and compared the DGGE finger- The present results showed that each patient harbored
printing patterns of these 2 clinical conditions. a unique pericoronitis bacterial community with a few
Although molecular fingerprinting does not allow im- dominant species (represented by more intense
mediate discrimination among bacterial species, it bands). However, some bands were shared across
does show the relevant difference in the predominant many DGGE profiles. These bands were intense in
bacterial composition and thus facilitates direct com- some cases and faint in others, indicating there were
parisons of microbial communities from different sam- no dominant species across samples.
ples of interest.6 In addition, DGGE-generated DGGE profiling of bacterial populations in the peri-
molecular fingerprinting allows the study of changes coronal pockets of patients in the P group displayed
in individual microbial communities over time.21 Bac- slightly less diversity than those of patients in the
terial DNA profiles determined by the DGGE method ASY control group. This finding suggests that
have shown dominant phylotypes in a community, pericoronitis-associated microbiota become less
indicating the suitability of this method for studying diverse, perhaps because certain groups of microbes
overall microbial diversity in environmental samples. supplant or dominate the biofilm as pericoronitis pro-
This method also has proved useful for exploring bac- gresses. One possible explanation for this difference in
terial communities in arthropods.22 microbial diversity is that larger proportions of
DGGE analyses were performed based on the posi- pericoronitis-associated bacteria are found in symp-
tion and intensity of each detected band. Normalized tomatic than in asymptomatic pericoronitis cases, as
banding patterns were used to generate a similarity co- some research articles have reported.26 Another expla-
efficient (Cs; range, 0 to 100%) for comparisons be- nation for the lower diversity is that symptomatic peri-
tween samples. Clustering techniques were applied coronitis pockets create more retentive niches for
to the DGGE profiles to identify samples with similar pericoronitis-associated micro-organisms, which in-
patterns. The resulting dendrogram showed that pro- crease their total numbers but subsequently decrease
files from pericoronitis samples were clustered ac- the richness of the bacterial community.
cording to the presence or absence of symptoms, The present study showed that DGGE analysis is use-
indicating that the bacterial communities associated ful for the assessment of the diversity of the pericoronitis
with these 2 clinical situations were different. The microbiota and for rapid comparison of microbial
ZHANG, SUN, AND YANG 489

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