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Preliminary Molecular Analysis of Bacterial Composition in Periapical Lesions with Primary Endodontic
Infections of Deciduous Teeth
1
Background
The bacterial composition of periapical lesions in deciduous teeth has not been well
documented. This study was designed to explore the bacterial compositions, especially the dominant
bacteria in periapical lesions using 16S rRNA sequencing.
Methods
Tissue samples were collected from 11 periapical lesions in deciduous teeth with primary
endodontic infections. DNA was extracted from each sample and analyzed using 16S rRNA cloning and
sequencing for the identification of bacteria.
Results
All DNA samples were positive for 16S rRNA gene PCR. One hundred fifty-one phylotypes
from 810 clones were identified to belong to eight phyla, and each sample contained an average of 25.9
phylotypes. In addition, 59 phylotypes were detected in more than two samples, and Fusobacterium
nucleatum (8/11), Dialister invisus (8/11), Campylobacter gracilis (7/11), Escherichia coli DH1 (6/11),
Aggregatibacter segnis (6/11), and Streptococcus mitis (6/11) were the most prevalent species.
Furthermore, 45 as-yet-uncultivated phylotypes were also identified.
Conclusion
Chronic periapical lesions in deciduous teeth contained polymicrobial infections. F.
nucleatum, D. invisus, C. gracilis, E. coli DH1, A. segnis, and S. mitis were the most prevalent species
detected by 16S rRNA sequencing.
Keywords: 16S rRNA sequencing; bacteria; deciduous teeth; periapical lesions
2
Chronic periapical periodontitis is the major cause of premature loss of deciduous teeth which believed to
be very harmful to children. In some cases, it could even cause permanent hypoplastic teeth1. It is widely
accepted that chronic periapical periodontitis is caused by endodontic infection2. For a long time, it had
been believed that bacteria were not able to inhabit in these lesions at chronic phase3 and the expansion of
periapical lesions was caused by local immune responses (type III and IV allergy). However, recent
studies of extraradicular endodontic infection reported that bacteria have been detected in periapical
lesions4,5,6. Bacteria in periapical lesions can cause hyperemia, edema of the periodontal ligament,
extravasation of neutrophilsm, activation of osteoclasts, and eventually osteoclastic destruction of the
bones surrounding the periapex through inflammation7,8. Therefore, eliminating infection by
antimicrobial treatment is the most effective strategy of periapical periodontitis. Consequently the choice
of antimicrobial treatment for periapical periodontitis should be based on the most probable
microorganisms and susceptibility testing. Based on those informations, researches on detection of the
bacterial compositions in periapical lesions of deciduous teeth are important and necessary for developing
more effective strategies. However, literature concerning the periapical bacterial analysis of deciduous
teeth is limited, and studies on periapical lesion tissues bacterial flora are even less.
Traditionally, endodontic infections have been studied by culture-dependent methods. Nonviable,
uncultivable, slow-growing, or fastidious bacteria contribute to underestimation of microorganisms with
culture methods. Studies reported that approximately 50% of oral bacteria are unculturable9. In contrast to
conventional culture methods, molecular techniques have advantage of often detecting uncultivable or
difficult to grow bacteria. However using specific DNA probes can only detect certain target bacteria; it is
not effective towards unknown bacteria. As an alternative, direct amplification of 16S rRNA genes from
DNA, followed by cloning and sequencing is the more advanced and reliable study method to detect the
macro flora in the environment for determining bacteria diversity. It has assisted in identifying previously
3
uncharacterized bacteria with small sample sets in root canal infections10,11.
Therefore, our study aimed to investigate the bacterial compositions including unculturable or fastidious
bacteria in periapical lesions associated with primary endodontic infections of deciduous teeth using 16S
rRNA sequencing.
METHODS
Patient population and clinical specimens
All clinical procedures performed in this study were approved by the Human Volunteers Research and
Ethics Committee of Capital Medical University School of Stomatology, Beijing, China. All procedures
were explained in advance to the participating parents, and parental informed consent was obtained from
all patients.
Test samples were collected from 11 patients (5 boys and 6 girls, age range 5- 8 years). The patients were
referred to dental treatments at the Department of Pediatric Dentistry, Capital Medical University School
of Stomatology, Beijing, China. The primary molars, diagnosed with chronic periapical periodontitis
according to the criteria established by Torabinejad and Walton12 and without previous dental treatment,
which had to be extracted, were selected in the study. All extracted molars were at root stable period. No
apparent communication between the periodontal pockets and the periapical lesions were detected by
periodontal probe. Patients who presented with systemic diseases, and teeth presenting with sinus tracts,
periodontal probing depth more than 3 mm, or had received antibiotic therapy within 3 months prior to
collection, were excluded from the study.
Sampling procedure
After local anesthesia, the tooth and its surrounding area were thoroughly swabbed with sterile gauze
4
soaked in 0.2% chlorhexidine gluconate solution. During the tooth extraction, the tongue and lips were
held back, and the suction apparatus was carefully performed to avoid salivary contamination. The
periapical tissue was collected from both the socket and furcation of the primary molar by sterile curette
after extraction and immediately washed with sterile saline to remove residual blood and planktonic
bacteria. All periapical tissue samples were divided into 2 segments. One of them was sent to
histopathological examination. The other one was immediately stored in cryotube containing TE buffer
(10 mmol/L Tris-Hcl, 1 mmol/L EDTA, PH7.6) and frozen at -80˚C for DNA extraction.
DNA extraction and Amplification of 16S rRNA genes
Tissue samples were pelleted by centrifugation, and the total bacterial genomic DNA was extracted using
the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA) according the
manufacturer’s instruction. All DNA samples were kept at -20 °C until further analysis. The 16S rRNA
genes were amplified using universal primers 27F (5’-AGA GTT TGA TC[A/C] TGG CTC AG-3’) and
1492R (5’-TAC GG[C/T] TAC CTT GTT ACG ACT T-3’)10. All PCR reactions were carried out in the
GeneAmp PCR System 9700 (Applied Biosystems, USA) with 50 μL reaction mixtures containing 100
ng total bacterial DNA, 200 μmol/L of each deoxynucleoside triphosphate, 1 μM of each primer, 5 μL 10x
PCR buffer, 2 mM MgCl2, and 1.25 U Taq DNA polymerase (all reagents purchased from Clontech,
USA). The thermocycling program included an initial denaturation step at 95 °C for 5 min, followed by
35 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and extension at 72 °C for 1.5
min and a final extension step at 72 °C for 10 min. The negative control contained sterile water. PCR
products were examined using 1.5% agarose gel electrophoresis. Amplification products (approximately
1500 bp in length) were purified with the EZNATM Ultra-Sep Gel Extraction kit (Omega Biotec, USA)
following the manufacturer’s instruction.
5
Clone library sequencing
Purified amplicons were ligated into the pGEM-T EasyVector (Promega Corporation, Madison, WI) and
then transferred into E. coli competent cells of JM109 strain (Promega, USA) in accordance with the
manufacturer’s instructions. The Transformed cells were then plated onto Luria-Bertani agar plates
supplemented with ampicillin and incubated at 37 °C overnight. Eighty colonies were randomly selected
for each sample, and all colonies were analyzed when less than eighty. The insertion of amplicons was
determined by PCR using vector primers T7 (5’-TAA TAC GAC TCA CTA TAG GG-3’) and SP6
(5’-ATT TAG GTG ACA CTA TAG AAT-3’). Clones without inserts were excluded from further analyses.
Plasmid DNA from recombinant clones was purified using the Qiagen Plasmid Purification Kit (Qiagen,
UK) and sequenced with the ABI Prism cycle sequencing kit (Applied Biosystems) and universal 27F
primer. The clone libraries were checked for the presence of chimera
using the Chimera-Check
program13. The nucleotide sequences were analyzed and matched using the program Sequence Match at
the Ribosomal Database Project II and by BLASTN (BLAST 2.0; http://www.ncbi.nlm.nih.gov/blast) to
determine the highest identity to known sequences in the database, the clone sequences that had 98-100%
identity with the same GenBank sequence were considered as from the same species10.
RESULTS
11 samples were histopathologically in accordance with chronic periapical periodontitis diagnostic
criteria in this study.
All DNA samples were tested positive by PCR amplification using the universal 16S rRNA primer,
6
compared with the negative control’s zero yield of amplicons. Overall, 151 different phylotypes were
identified from 814 sequenced clones. Four chimeric sequences were excluded from subsequent analyses.
The remaining 810 sequences were identified by alignments with related known sequences in GenBank.
The final number of clones sequenced per sample is shown in supplement material.
The sampled bacteria belonged to eight phyla (Figure 1), with Firmicutes as the dominant one that
accounted for 48.3% of the phylotypes and 43.0% of the identified clones (73 phylotypes, 348 clones).
The mean number of bacterial taxa per sample was 25.9 with a range of 17-31, and 59 phylotypes were
detected in more than two samples. Fusobacterium nucleatum and Dialister invisus were the most
prevalent species (8/11), followed by Campylobacter gracilis (7/11), Escherichia. coli DH1 (6/11),
Aggregatibacter segnis (6/11), and Streptococcus mitis (6/11) (Table 1). In addition，Enterococcus
faecalis, was also identified in 3 out of 11 cases. Furthermore, 45 as-yet-uncultivated phylotypes were
identified.
DISCUSSION
In our study, we investigated the bacterial composition of 11 periapical lesions of deciduous teeth
associated with primary endodontic infections. Our results showed that all samples contained bacteria,
which is similar to the findings from studies on permanent teeth14,15. Thus, our study provided additional
information on the microbial composition in periapical lesions of deciduous teeth. And it could be useful
for requiring a more effective treatment in periapical periodontitis in children. Meanwhile, it was also
confirmed that 16S rRNA sequencing using in our study was much higher sensitivity than the culture
methods16,17,18,19. This finding suggests that molecular method can be a preferred choice of analysis for
periodontal pathogens.
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In this study, as well as in other studies17,20,21, the phylum Firmicutes showed the largest number of
species. As the most detected species, Fusobacterium nucleatum was detected consistently in 8/11
(72.73%) of samples. F. nucleatum is a gram-negative, non-spore-forming, nonmotile, obligatory
anaerobic rod bacterium, and it is usually isolated from infected root canal and periapical lesions with
high prevalence18,22,23,24. Cogulu et al. also reported that F. nucleatum could found both in endodontic
infections of deciduous and permanent teeth25. F. nucleatum is associated with severe tissue infections24
by adhering to and invading oral epithelial and KB cells to cause cells to produce a large amount of IL-6,
IL-8 and other inflammatory factors26,27. Another report showed that F. nucleatum was predominantly
detected among 58 clones from 6 infected root canals in single rooted teeth with periapical lesions before
treatment, whereas disappeared after treatment28. Based on accumulated information, it suggests that F.
nucleatum might play an important pathogenic role in periodontal diseases in deciduous teeth.
Dialister invisus and Campylobacter gracilis were another two frequently detected species in our study.
D. invisus is an oral anaerobic gram-negative coccobacillus. And Campylobacter gracilis is a nonmotile,
nonsporing, anaerobic, and gram-negative rod-shaped bacterium. Previous studies reported that D. invisus
was the most detected bacterium from the root canals with primary and secondary periapical
infections29,30,31, and C. gracilis can also be found with relatively high prevalence in those infections32. In
addition, they were both detected in persistent periapical lesions14,17,33. However, the pathogenicity of D.
invisus and C. gracilis are not yet well understood, and more investigations are required to reveal its role
in the persistence of periapical infections
As a member of the Pasteurellaceae family, Aggregatibacter is a genus of gram-negative, non-motile,
facultatively anaerobic rod bacteria. The growth of Aggregatibacter is mesophilic, but some species in
this genus, including Aggregatibacter segnis, are capnophilic. A. segnis is a regular member of the human
8
oral flora, particularly in dental plaques, and it can be isolated from the pharynx as well. It has also been
occasionally isolated from human infections including infective endocarditis34. Due to the very limited
focus on A. segnis, more detailed studies are clearly required to further explore this bacterium’s potential
pathogenicity.
The most commonly isolated gram-positive cocci were Streptococcus spp., the members of which are the
most frequently identified bacteria in teeth with periapical lesions. In accordance with many previous
studies9,14,15,16,17,18, the high occurrence of streptococci was found in infected root canals and closed
periapical lesions in permanent teeth. As well, streptococci were detected in 85% infected root canals
with periapical lesions of deciduous teeth35. Thus, streptococci might play a certain role not only in
infected root canals but also in periapical lesions in deciduous teeth. Moreover, E. faecalis, the other
gram-positive cocci, was also identified in 3 out of 11 cases. It has been commonly believed that the
presence of E. faecalis in untreated patients was very rare. However, studies suggested that this specie
was an important agent in endodontic failure and was frequently encountered in root-filled teeth with
periapical lesions36, 37. Evidence has shown that E. faecalis may contribute to the persistent periapical
infections by both biofilm formation and soft-tissue adhesion17. Cogulu et al.25 reported that E. faecalis
was highly associated with periapical radiolucency and with previous pains in both deciduous and
permanent teeth of primary infection. Though questions remain in whether E. faecalis plays an important
role in the pathogenesis of recalcitrant periapical diseases, in our result implying that E. faecali might
involve in severe periapical infections of deciduous teeth.
In the present study, some gram-negative anaerobic species that were commonly found in primary
endodontic infections, such as P. gingivalis, P. intermedia, P.nigrescens, were frequently detected in
9
periapical lesions. At the same time, many fastidious bacterial species were detected in the periapical
lesions of deciduous teeth by molecular methods without culturing. Among them, two fastidious
anaerobic bacteria, Filifactor alocis and Treponema sp., were detected in 3/11 (27.27%) and 2/11 (18.18%)
of cases, respectively. In addition, 45 as-yet-uncultivated phylotypes were also identified. This is an
important finding suggesting previously unknown and uncultivated bacteria may have roles in the
pathogenesis of periapical diseases.
In summary, findings from this study support the belief that periapical lesions of deciduous teeth are
frequently correlated with mixed intraradicular infections, and Fusobacterium nucleatum, Dialister
invisus, Campylobacter gracilis, Escherichia coli DH1, Aggregatibacter segnis, and Streptococcus mitis
were common members of the microbiota. We also confirmed the microbial diversity of periapical lesions
and need to enhance the ongoing characterization of periapical bacteria for deciduous teeth. Although the
subject population was small, our study will help to better understand the roles of those aforementioned
bacteria in the process of periapical periodontitis. It is beneficial to fully comprehend the pathogenesis of
apical periodontitis and hence lead to develop more effective therapeutic strategies for periapical
periodontitis in deciduous teeth. In addition, the more effective strategies for periapical infection
treatment might due to reserved more diseased infected teeth.
10
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14
Figure. 1. Diversity of phylotypes in periapical lesions with primary endodontic infections of deciduous
teeth
15
Table 1. Prevalence of bacterial species identified in 11 extraradicular samples
Sample
Clone
Porportion
Bacteria taxa
No.
Porportion
No.
(%of total)
(%of total)
Fusobacterium nucleatum
8
72.73
31
3.83
Dialister invisus
8
72.73
12
1.48
Campylobacter gracilis
7
63.64
62
7.65
Escherichia coli DH1(ME8569)
6
54.55
23
2.84
Aggregatibacter segnis
6
54.55
15
1.85
Streptococcus mitis
6
54.55
8
0.99
Cardiobacterium hominis
5
45.45
25
3.09
Uncultured Veillonella sp. clone 7d17260
5
45.45
13
1.60
Anaeroglobus geminates
5
45.45
11
1.36
Streptococcus sanguinis
5
45.45
9
1.11
Veillonella sp. oral clone VeillF12
5
45.45
9
1.11
Campylobacter rectus
4
36.36
35
4.32
Selenomonas sputigena
4
36.36
9
1.11
Streptococcus cristatus
4
36.36
8
0.99
Streptococcus oralis
4
36.36
7
0.86
Uncultured bacterium clone H2-plate10_B02
4
36.36
7
0.86
Prevotella nigrescens
4
36.36
7
0.86
Streptococcus mutans
4
36.36
6
0.74
Parvimonas micra
3
27.27
33
4.07
16
Capnocytophaga granulose
3
27.27
21
2.59
Methylobacterium fujisawaense
3
27.27
19
2.35
Dialister pneumosintes
3
27.27
18
2.22
Filifactor alocis
3
27.27
16
1.98
Haemophilus sp. clone ABLBa33
3
27.27
11
1.36
streptococcus sp. oral taxon 058
3
27.27
10
1.23
Terrahaemophilus aromaticivorans
3
27.27
10
1.23
Streptococcus intermedius
3
27.27
9
1.11
Solobacterium moorei
3
27.27
9
1.11
Enterococcus faecalis
3
27.27
9
1.11
Capnocytophaga gingivalis
3
27.27
8
0.99
Selenomonas infelix
3
27.27
6
0.74
Oribacterium sp. oral taxon 078 clone _X095
3
27.27
6
0.74
Eubacterium brachy cloneWWP_SS3_P14
3
27.27
5
0.62
Haemophilus parainfluenzae
3
27.27
5
0.62
Streptococcus anginosus
3
27.27
4
0.49
Prevotella intermedia
2
18.18
22
2.71
Cardiobacterium valvarum
2
18.18
13
1.60
Lactobacillus gasseri
2
18.18
10
1.23
Uncultured Veillonella sp. clone KLONG06
2
18.18
9
1.11
Campylobacter concisus
2
18.18
9
1.11
Bacteroidales genomosp
2
18.18
8
0.99
Gemella morbillorum
2
18.18
6
0.74
17
Anoxybacillus flavithermus
2
18.18
6
0.74
ncultured Dialister sp. clone 7BB286
2
18.18
6
0.74
Veillonellaceae bacterium oral clone _K024
2
18.18
5
0.62
Escherichia fergusonii
2
18.18
5
0.62
Prevotella denticola
2
18.18
5
0.62
Prevotella oris
2
18.18
5
0.62
601G07(oral)
2
18.18
4
0.49
Catonella morbid
2
18.18
3
0.37
Selenomonas sp. oral clone DO042
2
18.18
3
0.37
Haemophilus haemolyticus
2
18.18
3
0.37
Cronobacter sakazakii
2
18.18
3
0.37
Porphyromonas gingivalis
2
18.18
3
0.37
Mogibacterium diversum
2
18.18
2
0.25
Kingella oralis
2
18.18
2
0.25
Leptotrichia buccalis
2
18.18
2
0.25
Treponema sp. oral taxon 270
2
18.18
2
0.25
Rothia dentocariosa
2
18.18
2
0.25
uncultured Porphyromonadaceae bacterium clone
18