Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis.

doi:10.1111/iej.12361

Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis

M. N. Zakaria1,2,3, T. Takeshita1, Y. Shibata1, H. Maeda2, N. Wada2, A. Akamine2 & Y. Yamashita1 1

Section of Preventive and Public Health Dentistry, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Fukuoka; 2Division of Oral Rehabilitation, Department of Endodontology and Operative Dentistry, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; and 3Faculty of Medicine, Jenderal Achmad Yani University, Bandung, Indonesia

Abstract Zakaria MN, Takeshita T, Shibata Y, Maeda H, Wada N, Akamine A, Yamashita Y. Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis. International Endodontic Journal.

Aim To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. Methodology Apical lesion samples were collected from 12 patients (23–80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. Results Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacteri-

um acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. Conclusion Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species. Keywords: 16S rRNA, apical periodontitis, clone library. Received 7 March 2014; accepted 31 July 2014

Introduction Correspondence: Yoshihisa Yamashita, Section of Preventive and Public Health Dentistry, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (tel: +81 (92) 642-6353; fax: +81 (92) 642-6354; e-mail: [email protected]).

© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd

Microbial factors are considered the major cause of persistent inflammation after root canal treatment (Nair 2004). Bacteria that are either resistant or inaccessible to root canal treatment persist and facilitate ongoing infection (Siqueira & R^ ocas 2008). Numerous studies have examined the presence of microbes in

International Endodontic Journal

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Microbial community in persistent apical periodontitis Zakaria et al.

the root canal (Waltimo et al. 1997, Molander et al. 1998, Sundqvist et al. 1998, Siqueira & R^ ocas 2004, Zhu et al. 2010, Wang et al. 2012). Common species that are frequently found in root filled teeth are Enterococcus faecalis and Candida albicans (Waltimo et al. 1997, 2003, Love 2001, Peciuliene et al. 2001, Pinheiro et al. 2003, Stuart et al. 2006, Zoletti et al. 2006, Zhu et al. 2010). Therefore, many studies have focused on developing effective strategies to eradicate them from the root canal (Ates et al. 2005, Ercan et al. 2006, Baca et al. 2011, Tong et al. 2013). Bacterial survival is closely related to their ability to adapt to hostile environments and biofilm formation is considered an effective survival strategy and a common cause of persistent infection (Chavez de Paz 2007). As a result of continuous irritation by microorganisms in the root canal, the immune response creates a barrier to prevent further bacterial spread. In the periapical area, the bone structure can be resorbed and replaced by fibrous connective tissue (granulation tissue) containing defence elements, such as immune cells and molecules (Saber et al. 2012). These barriers limit the egress of the microorganisms, although microbial products can diffuse through and induce periradicular pathosis. Some surviving microorganisms might also have the ability to invade and overcome host tissue defences, enabling persistent infection of the periapical area. Previous studies have examined biofilm formation in the canal space or on the outer surface of the apical portion of the root (Su et al. 2010, Wang et al. 2012). However, information on extraradicular infection resulting in a persistent lesion is limited and is mostly associated with Actinomyces or Propionibacterium species (Sunde et al. 2002, Siqueira & R^ ocas 2003, Ricucci & Siqueira 2008). Difficult-to-culture bacteria are often missed using traditional culture methods, resulting in an underestimation of the bacterial diversity of the community associated with persistent disease. Given that apical periodontitis has a heterogeneous aetiology with multiple species implicated, molecular genetics was used to identify the microbial community (bacteria and C. albicans) invading the periapical lesion in persistent apical periodontitis.

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radiographic evidence of periradicular lesions, who visited the Endodontic and Operative Dentistry Clinic at Kyushu University Hospital for apicectomy following the diagnosis of persistent apical periodontitis (Table 1). The diagnosis was based on a history of persistent lesions despite adequate root filling or no improvement in clinical signs after root canal retreatment, as described in Table 1. Radiographs of the periapical lesions are shown in Table 1. Patients with spontaneous pain or pain on percussion or pain upon palpation were considered symptomatic, whilst patients with none of these conditions were defined as asymptomatic. The Ethics Committee of Kyushu University Faculty of Dental Science approved this study and written informed consent for the procedures was obtained from all patients. Root fracture and deep periodontal pockets (>3 mm) were not observed in any subject. Patients who had received antibiotic therapy within the 2 months before surgery and those with chronic systemic diseases or immunodeficiency disorders were excluded. Apical lesion samples were collected during apicectomy using standard procedures and aseptic techniques. Patients used an oral rinse of 0.2% chlorhexidine before surgery and the surgical site was disinfected with 0.2% chlorhexidine. A full-thickness submarginal flap was raised and after bone removal, the tissue was enucleated prior to root end resection and placed in a sterile vial containing 5 mL of PBS. Then, the specimen was immediately transferred to the laboratory for DNA extraction. Saliva from each patient was collected as a control to ensure that saliva contamination was avoided during sampling.

Terminal restriction fragment length polymorphism analysis

Materials and methods

DNA was extracted from the lesion specimen and saliva, as described previously (Takeshita et al. 2007). To compare the bacterial composition between the lesion specimen and saliva, T-RFLP analyses of the 16S rRNA gene were performed, as described previously (Takeshita et al. 2009). The 6-FAM labeled T-RFLP profiles were aligned and hierarchical cluster analysis was performed using a correlation distance metric and Ward’s method to identify distinct T-RFLP profiles in the lesion specimens and saliva.

Patients and samples

Clone library analysis

In all, 12 periapical lesion samples were collected from 12 patients from 23 to 80 years of age with

Twelve apical lesion samples were cloned and sequenced. Polymerase chain reaction (PCR) of the



Zakaria et al. Microbial community in persistent apical periodontitis

Table 1 Characteristics of patients and apical lesions Patient characteristics

Sample No.

Gender

Age (years old)

Tooth number

Follow-up perioda

Apical lesions

Conventional retreatment

Sinus tract

Symptomb

Radiolucency size (mm)

Number of bacteria (cells)

G1

Female

62

16

1 year

+

4.1

2.5 9 10

G2

Female

72

22

5 years

+

4.7

9.8 9 104

G3

Female

42

11.21

20 years

11: 2.3 21: 3.0

9.6 9 103

G4

Female

58

21

1.5 years

+

5.1

5.1 9 103

G5

Female

67

15

15 years

+

5.7

8.1 9 104

G6

Female

25

11

2 years

+ 1 monthc

3.5

1.3 9 105

G7

Female

80

34

30 years

+ 2 monthsc

+

7.2

4.9 9 104

G8

Female

52

17

8 years

+ 3 monthsc

+

+

4.9

2.8 9 104

G9

Female

23

12

6 years

+ 3 monthsc

+

+

11.0

1.1 9 104

+ 4 monthsc

+

+


Radiograph

5


3


Table 1 Continued Patient characteristics

Sample No.

Gender

Age (years old)

Tooth number

Follow-up perioda

Apical lesions

Conventional retreatment

Sinus tract

Symptomb

Radiolucency size (mm)

Number of bacteria (cells)

G 10

Female

52

12

20 years

3.0

6.2 9 10

G11

Male

65

44

12 years

4.5

3.1 9 105

G12

Male

32

42

13 years

3.2

1.9 9 106

+

Radiograph

4

a

Period from the first initial endodontic treatment. Symptoms include any sign of spontaneous pain, pain to percussion or pain upon palpation. Conventional retreatment was carried out for the indicated months, but pain or pus discharge did not disappear until the end of the treatment. b c

16S rRNA fragment was conducted using the universal primer 8F with a 15-bp adaptor sequence for cloning (50 -GAA TTC CTG CAG CCC AGA GTT TGA TYM TGG CTC AG-30 ) and 1492R with a 15-bp adaptor sequence (50 -ACT AGT GGA TCC CCC GGY TAC CTT GTT ACG ACT T-30 ) and PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies, Palo Alto, CA, USA) in 25 lL of PCR mixture, according to the manufacturer’s instructions. After an initial 2 min at 95 °C, then 30 cycles of 30 s at 95 °C, 30 s at 60 °C and 2 min at 65 °C were performed. The PCR products were gel-purified using Wizard SV Gel and the PCR Clean-up System (Promega, Madison, WI, USA). The purified PCR products were inserted into pBluescript II SK (+) vector (Stratagene, La Jolla, CA, USA) using an In-Fusion HD Cloning Kit (Clontech Laboratories, Mountain View, CA, USA) and transformed into Escherichia coli DH5a. The transformed bacteria were plated onto Plusgrow (Nacalai-Tesque, Kyoto, Japan) nutrient agar supplemented with ampicillin (100 lL mL1) and incubated overnight at 37 °C. The nucleotide sequences of the inserts were determined using a Big Dye Terminator Cycle Kit (Applied Biosystems, Foster City, CA, USA) and an M13F

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primer (50 -GGT TTT CCC AGT CAC GAC GTT-30 ) in a genetic analyzer (ABI PRISM 3730 Genetic Analyzer, Applied Biosystems). For each sequence, the nearestneighbour species with >98.5% identity were first identified using BLAST against 778 oral bacterial 16S rRNA gene sequences (HOMD 16S rRNA RefSeq, ver. 11.0) from the Human Oral Microbiome Database (http://www.homd.org). Sequences with no matches were compared against the database Nucleotide collection (nr/nt) of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/).

Quantitative and qualitative PCR analysis To compare the total bacteria per sample, quantitative real-time PCR was performed using a QuantiFast SYBR Green PCR kit (Qiagen, Hilden, Germany) in a StepOne Real-Time PCR System (Applied Biosystems), according to the manufacturers’ instructions. The universal bacterial primers 806F (50 -TTA GAT ACC CYG GTA GTC C-30 ) and 926R (50 -CCG TCA ATT YCT TTG AGT TT-30 ) were used. The relative amounts of total bacteria were calculated using the modified comparative Ct method using DNA extracted from Streptococcus mutans Xc as a real-time PCR



standard. Quantification of the number of bacterial cells in the sample was performed based on a comparison of the Ct value of the sample with a standard curve generated from serial dilutions of S. mutans chromosomal DNA extracted from a known number of cells and their Ct values. One CFU of S. mutans was defined to be one cell of the organism. The fimA genotype was evaluated using a nested PCR approach. First, PCR was carried out using a pair of Porphyromonas gingivalis universal M11 primers (50 -AAT CTG CGA CGC TAT-30 ) and type I fimAR (50 -AAC CCC GCT CCC TGT ATT CCG A-30 ) (Amano et al. 1999) with PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies), according to the manufacturer’s instructions. The temperature conditions were 2 min at 95 °C for denaturation, followed by 30 cycles at 95 °C for 30 s, 2 min at 62 °C and 2 min at 65 °C. The second PCR step utilized pairs of specific fimA gene primers to determine the genotype, as shown in Table 2. The annealing temperature used was 60 °C for fimA types I, III, and V and 57 °C for types II and IV. The absence of E. faecalis was confirmed using a nested PCR approach. Amplicons of the 16S rRNA gene used for cloning and sequencing analysis were diluted 20-fold and specific primers for E. faecalis were used for the second round of PCR with the same polymerase as previously (Table 2). To detect C. albicans, nested PCR was performed using fungal universal primers (ITS1-ITS4) for the first round of PCR and primers specific for C. albicans for the second round (Table 2). The amplicons from

the first round of PCR were diluted 20-fold before the second PCR with the specific primers.

Results Apical lesions were obtained from the persistent periapical lesions of 12 subjects. Seven samples were collected from symptomatic lesions and five were collected from asymptomatic lesions. The radiographic findings varied from a widened periodontal ligament to alveolar bone destruction. The characteristics and clinical findings of the study patients are summarized in Table 1.

Bacterial composition in apical lesion samples The PCR assay using universal bacterial primers of the 16S rRNA gene showed bacteria in every sample. Quantitative PCR analysis of the total bacteria identified 5.1 9 103 to 1.9 9 106 cells (mean SD; 2.0 5.2 9 105) in the lesion samples. The nucleotide sequences of the 16S rRNA genes (about 600 bp) of 1040 clones were determined and these corresponded to 99 bacterial taxa, of which 62 were detected in only one sample each. The mean number of taxa detected was 15 7 (range 3–26) per sample. The bacterial composition varied amongst the samples (Fig. 1); all of the identified bacterial taxa are listed in Table 3. The dominant species were P. gingivalis (19.8% of the clones), Propionibacterium acnes (17.2%), Fusobacterium nucleatum (11.3%),

Table 2 Specific fimA gene primers and species-specific primers used in the study Primer target Type I fimA Type II fimA Type III fimA Type IV fimA Type V fimA Enterococcus faecalis ITS1-F ITS4 Candida albicans

Sequence

Size (bp)

CTG TGT GTT TAT GGC AAA CTT C AAC CCC GCT CCC TGT ATT CCG A ACA ACT ATA CTT ATG ACA ATG G AAC CCC GCT CCC TGT ATT CCG A ATT ACA CCT ACA CAG GTG AGG C AAC CCC GCT CCC TGT ATT CCG A CTA TTC AGG TGC TAT TAC CCA A AAC CCC GCT CCC TGT ATT CCG A AAC AAC AGT CTC CTT GAC AGT G TAT TGG GGG TCG AAC GTT ACT GTC GTT TAT GCA TGG CAT AAG AG CCG TCA GGG GAC GTT CAG CTT GGT CAT TTA GAG GAA GTA A TCC TCC GCT TAT TGA TAT GC TTT ATC AAC TTG TCA CAC CAG A ATC CCG CCT TAC CAC TAC CG

392

Amano et al. (1999)

257

Amano et al. (1999)

247

Amano et al. (1999)

251

Amano et al. (1999)

462

Nakagawa et al. (2000)

310

Zoletti et al. (2006)


Ref

Li et al. (2012) 273

Luo et al. (2002)


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Table 3 Bacteria taxa detected in all apical lesion samples Number of clones in each samplea

6

No.

Bacterial taxa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

Abiotrophia defective Acinetobacter baumannii Acinetobacter johnsonii Acinetobacter schindleri Actinomyces cardiffensis Actinomyces dentalis Actinomyces gerencseriae Actinomyces graevenitzii Actinomyces massiliensis Actinomyces sp. HOT-169 Actinomyces sp. HOT-180 Actinomyces sp. HOT-525 Actinomyces sp. HOT-877 Afipia sp. HOT-652 Alloprevotella sp. HOT-308 Bacteroidales sp. HOT-274 Bergeriella denitrificans Burkholderia cepacia Campylobacter gracilis Campylobacter rectus Corynebacterium kroppenstedtii Dialister invisus Dialister pneumosintes Enterococcus saccharolyticus Eubacterium nodatum Filifactor alocis Finegoldia magna Fretibacterium fastidiosum Fretibacterium sp. HOT-359 Fusobacterium nucleatum Gemella haemolysans Gardnerella vaginalis Granulicatella elegans Haemophilus parainfluenzae Lachnospiraceae sp. HOT-373 Lactobacillus gasseri Lactobacillus plantarum Lactobacillus salivarius Massilia niastensis Massilia timonae Mogibacterium timidum Neisseria elongata Neisseria flava Neisseria flavescens Neisseria subflava Neisseria sp. HOT-020 Neisseria sicca Olsenella uli Parvimonas sp. HOT-110 Parvimonas micra Peptostreptococcaceae sp. HOT-113 Peptostreptococcus stomatis Porphyromonas endodontalis Porphyromonas gingivalis Prevotell intermedia


G1

G2

G3

G4

G5

G6

G7

G8

G9

G11

G12

1 17 1

1 5 1

G10

1 3 3

1 3 2

1

1

1 1 3

1 2

2

1 1 1 1 1 2

4 1

1

3 1

1

1

1

3

1

1

2

10

69

8

1 6 4

1

1

2 2 5

1 1 15

1

27

4 10

5 1

4

2 5 6

6

1 6 9 2 2 1 3 1 2 23

2

12

5 10

2 1 13

5 9

2 14 1 1 16

9

14

16 2

1 1

12 1

43

91

34

Total (%) 1 18 1 5 2 3 3 1 3 4 1 1 4 4 1 1 1 1 1 6 1 6 1 1 11 4 1 5 1 118 40 7 5 12 1 6 9 2 2 1 33 1 2 37 5 12 1 18 99 2 44 3 2 206 1

(0.1) (1.7) (0.1) (0.5) (0.2) (0.3) (0.3) (0.1) (0.3) (0.4) (0.1) (0.1) (0.4) (0.4) (0.1) (0.1) (0.1) (0.1) (0.1) (0.6) (0.1) (0.6) (0.1) (0.1) (1.1) (0.4) (0.1) (0.5) (0.1) (11.3) (3.8) (0.7) (0.5) (1.1) (0.1) (0.6) (0.9) (0.2) (0.2) (0.1) (0.3) (0.1) (0.2) (2.6) (0.5) (1.1) (0.1) (1.7) (0.9) (0.2) (4.23) (0.3) (0.2) (19.8) (0.1)



Table 3 Continued Number of clones in each samplea No.

Bacterial taxa

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

Prevotella buccae Prevotella marshii Prevotella melaninogenica Prevotella nigrescens Prevotella oris Prevotella pleuritidis Prevotella sp. HOT-308 Prevotella sp. HOT-472 Propionibacterium acnes Propionibacterium granulosum Pseudomonas aeruginosa Ralstonia pickettii Rhodococcus fascians Roseomonas vinaceus Rothia aeria Rothia mucilaginosa Selenomonas infelix Slackia exigua Sneathia amnionii Staphylococcus warneri Staphylococcus epidermidis Staphylococcus hominis Streptococcus australis Streptococcus infantis Streptococcus intermedius Streptococcus mitis Streptococcus peroris Streptococcus pneumoniae Streptococcus sanguinis Streptococcus salivarius Streptococcus sp. HOT-431 Streptococcus sp. HOT-061 Streptococcus sp. HOT-064 Streptococcus sp. HOT-068 Streptococcus sp. HOT-071 Tannerella forsythia Terrahaemophilus aromaticivorans Treponema denticola Treponema maltophilum Treponema socranskii Treponema sp. HOT-268 Veillonella dispar Veillonella parvula Veillonella sp. HOT-158 Total number of colonies

a

G1

G2

G3

G4

G5

G6

G7

G8

G9

G10

G11

G12

2 1 2 4 2 2

4 1

3

1 1 14

1

9

9

58 3

88

3 4

1

2 1 1

1 4

3 1 3

1 2 1 1

1

1 1 2

1 1

1 1 2

1

2

4

39

1

1 1 3 1 1

10

3 5 18

1

1

1

16 4 8

1 1

3 1 2

2

1 1

1 1 1

8

5 1 1

79

93

95

68

98

93

85

1 91

1 1 90

59

93

96

Total (%) 2 1 2 8 6 2 1 1 179 3 3 7 1 1 1 8 2 4 1 5 13 1 4 7 19 44 2 1 1 23 4 10 1 3 1 5 1 1 1 14 1 2 1 1 1040

(0.2) (0.1) (0.2) (0.8) (0.6) (0.2) (0.1) (0.1) (17.2) (0.3) (0.3) (0.7) (0.1) (0.1) (0.1) (0.8) (0.2) (0.4) (0.1) (0.5) (1.2) (0.1) (0.4) (0.7) (1.8) (4.2) (0.2) (0.1) (0.1) (2.2) (0.4) (1) (0.1) (0.3) (0.1) (0.5) (0.1) (0.1) (0.1) (1.3) (0.1) (0.2) (0.1) (0.1)

Samples from symptomatic lesion was shown in bold.

Streptococcus mitis (4.2%) and Peptostreptococcaceae sp. HOT-113 (4.2%). F. nucleatum (8 of 12 subjects, 66.7%), P. gingivalis (7 of 12 subjects, 58.3%), and P. acnes (6 of 12 subjects, 50%) were detected frequently in the study population.


Enterococcus faecalis is associated with persistent endodontic infection, but it was not detected in any of the 12 samples using clone library analysis. The absence of E. faecalis was compared using nested PCR with sets of specific primers. The evaluation of


7


Figure 1 The dominant bacteria in each apical lesion sample. Taxa accounting for more than 10% of the total clones of each sample are shown.

C. albicans using nested PCR showed no detectable C. albicans in any sample. Porphyromonas gingivalis is detected in a high proportion in the community and was frequently detected in the subjects. It was hypothesized that its fimbria type contributes to its ability to invade periapical lesion, and determined the fimbria type of P. gingivalis using a nested PCR approach. Of the five fimbria types, only P. gingivalis with type I or IV fimA was found in the lesions. Figure 2 shows the results of the cluster analysis of T-RFLP patterns of the microbiota in apical lesions

Figure 2 Hierarchical cluster analysis of the terminal restriction fragment length polymorphism (T-RFLP) peak patterns of the microbiota in apical lesions and saliva samples. Correlation coefficients were used to define the distance between patterns, and Ward’s method was used for the distance function. G and S indicate samples from apical lesion and saliva, respectively.

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and saliva. The T-RFLP patterns of the saliva samples were clearly distinct from those of the apical lesions, except for the samples from G8 and G9.

Comparison of symptomatic and asymptomatic lesions The total bacterial amount in the apical lesion samples of the seven symptomatic cases was higher than in the five asymptomatic cases (3.3 7.0 9 105 vs. 1.1 1.0 9 105 cells), although the difference was not significant (P = 0.93, Mann–Whitney U-test). Significantly, more bacterial taxa were identified in the symptomatic group than in the asymptomatic group (19.1 6.3 vs. 9.4 5.0 per sample). Several bacterial species were detected only in the symptomatic group, such as Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua and Selenomonas infelix (Table 3). Notable bacterial combinations were found in the samples of subjects with symptomatic lesions (Fig. 1). The combination P. gingivalis, F. nucleatum and Peptostreptococcaceae sp. HOT-113 was identified in three of seven samples from symptomatic lesions. Interestingly, many P. gingivalis were also present in two asymptomatic samples (G6 and G7), whereas F. nucleatum was not detected in these samples. Streptococcus spp. occupied large proportions of the bacterial communities in two of the seven samples from symptomatic cases. P. acnes exceeded more than half of the clones identified in two samples (symptomatic and asymptomatic lesions) with



different combinations of bacterial species. Interestingly, P. gingivalis with type IV fimA was detected only in subjects with symptomatic lesions.

Discussion All of the samples in this study contained detectable bacteria. The molecular approach using the 16S rRNA gene with the clone library method can detect and identify even difficult-to-culture bacteria down to species level. A full-thickness submarginal flap was raised without involving the gingival sulcus to prevent contamination from the sulcular area. To exclude the possibility of bacterial contamination from saliva, the results of the lesions was compared with the corresponding saliva sample. The bacterial profiles differed completely between the apical region and saliva, except for two samples (G8 and G9). For these two samples, it was not possible to exclude the possibility of contamination by saliva during the apicectomy; however, these two samples were from the maxillary region where saliva control during surgery was more manageable. Therefore, it is also possible that bacteria in saliva invaded the lesions via sinus tracts. Another concern is the possibility of identifying dead bacteria when DNA derived from dead bacterial cells is amplified (Soejima et al. 2008). However, as inflammatory cells are striving to control the infection by phagocytosis, any dead bacteria tend to be digested and the DNA of the bacteria is released into the local environment, where it is likely to undergo spontaneous enzymatic decomposition rendering it undetectable in the amplification process (Brundin et al. 2010). A study of 36 periapical lesions using a culturebased method (Sunde et al. 2002) observed 67 species with an average of 4.1 2.5 microbes per sample; the dominant organism was Staphylococcus spp. (61.1%). In another study of 20 apical lesion samples (Fujii et al. 2009), the most commonly cultured species were P. acnes (16.2%), Staphylococcus epidermidis (9.5%) and Pseudomonas aeruginosa (6.8%). The only report using a cloning and sequencing method was the study by Handal et al. (2009), which excluded lesions with the presence of a sinus tract to eliminate possible communication with the oral cavity. They detected bacteria in 17 of 20 samples, whilst examining fewer clones in total (267 clones). In their study, the mean number of species per sample was seven and dominant species were Fusobacterium spp. (38%) and Prevotella spp. (35%). The bacterial flora


composition identified in the present study was more diverse as compared with previous studies, even though it was not possible to exclude the effect of including lesions with the presence of a sinus tract. In 13 periradicular lesions (2 asymptomatic and 11 symptomatic) analysed using pyrosequencing (Saber et al. 2012), only seven symptomatic lesions yielded PCR amplicons, in which Fusobacterium (21%), Streptococcus (8%), Prevotella (7.5%), Corynebacterium (7.2%) and Porphyromonas (6%) were most abundant. In this study, bacteria were detected in all samples. The bacterial populations across the lesions were diverse, but fewer bacteria were present in asymptomatic lesions than in symptomatic lesions. Fewer bacteria in the asymptomatic lesion might have precluded detecting bacteria in the study by Saber et al. (2012). As P. gingivalis and F. nucleatum were detected in biofilms formed in periapical lesions (Noguchi et al. 2005), the bacterial composition in the periapical lesions might also be related to the detachment of bacteria surviving in the form of a biofilm in the apical area, forming microcolonies within the body of the lesion. Alternatively, there might be the possibility that the detachment of biofilm occurred during the apicectomy and it is difficult to determine the exact derivation these bacteria in the lesion. Histological techniques with in situ hybridization without tissue destruction would be desirable in order to know the exact derivation of these bacteria. P. gingivalis enhances the ability of F. nucleatum to adhere to and invade epithelial cells (Han et al. 2000). Another study suggested that F. nucleatum enhances the attachment of P. gingivalis to human fibroblasts (Metzger et al. 2009). The synergistic interaction between P. gingivalis and F. nucleatum (Nagayama et al. 2001, Saito et al. 2008) and the growth of Veillonella sp. that was stimulated by F. nucleatum (Periasamy & Kolenbrander 2009) could explain the tendency of these bacteria to be found together in endodontic and periodontal infections. Propionibacterium acnes was detected in 6 of the 12 samples and comprised over half of the bacterial population in two samples (G11 & G12), suggesting that this species is involved in endodontic infections. An assessment of sodium hypochlorite and calcium hydroxide as antimicrobial agents in 24 necrotic root canals demonstrated that P. acnes and Streptococcus spp. were still found after 7 days of treatment, whereas the same treatment was very effective at controlling other bacterial taxa (R^ ocas & Siqueira 2011). P. acnes has also been found in the root canals


9


of endodontic infections (Sassone et al. 2008, Fujii et al. 2009). Another species of the same genus, Propionibacterium propionicum (formerly assigned to the genus Actinomyces), was more commonly detected (and is referred to as actinomycosis) (Siqueira & R^ ocas 2003, 2004, Ricucci & Siqueira 2008). Although considered an opportunistic pathogen mostly related to skin disease, P. acnes is believed to be involved in many infectious diseases of bones, joints, the eye and the brain, in addition to device-related infections (Perry & Lambert 2011). Interestingly, the dominant bacterial species in the periapical lesion in the present study differed from the dominant species found in the root canal in persistent periodontitis in previous studies (Peciuliene et al. 2001, Pinheiro et al. 2003, Siqueira & R^ ocas 2004, Wang et al. 2012). The distinct microbial composition from the root canal microbiota is likely due to different niche conditions amongst the sites. Anatomical attachment of the apical foramen to the periodontal ligament might allow bacteria that have the ability to invade the extraradicular tissue and possibly survive against host defences. A commonly isolated pathogen from therapy-resistant cases, E. faecalis, was not detected in any sequences from the clone library. As reported previously, E. faecalis possesses various survival and virulence factors and its prevalence in persistent infections ranges from 24% to 77% (Stuart et al. 2006). To confirm the absence of this microorganism, nested PCR using a set of primers specific for E. faecalis was performed, but it did not detect any PCR product in any of the samples. The difference in sampling site might explain the difference between the previous findings and this study, although most studies reporting the presence of E. faecalis isolated the microorganism from the root canals of treated teeth (Pinheiro et al. 2003, Zoletti et al. 2006, Zhu et al. 2010). Furthermore, C. albicans was also not detected using nested PCR with C. albicans-specific primers, suggesting that this species might not be able to survive in the extraradicular area. Porphyromonas gingivalis was detected in both symptomatic and asymptomatic persistent lesions. The virulence of P. gingivalis is likely due in part to its fimbriae, encoded by five different fimA genes, which are essential to its adherence and invasion ability (Amano et al. 1999, 2000). fimA type I is most prevalent in healthy individuals, whereas fimA type IV is often present in severe periodontitis (Amano et al. 2000). fimA type I was found only in asymptomatic

10


lesions and type IV only in symptomatic lesions, which is consistent with previous results. The involvement of Peptostreptococcaceae spp. and Prevotella spp. in symptomatic cases was consistent with a culture-based study that found a correlation between Peptostreptococcus ssp. and Fusobacterium in root canals that showed tenderness on percussion (Gomes et al. 1994) and Peptostreptococcus from root canals associated with clinical symptoms (Pinheiro et al. 2003). One sample from the present study that contained Peptostreptococcus stomatitis, Tannerella forsythia and abundant P. acnes clones was symptomatic, whereas another sample, which also contained many P. acnes clones but no bacteria suspected to be related to the symptoms, was asymptomatic. This observation suggested that abundant P. acnes clones might not be associated with symptoms. It seems logical to assume that specific bacteria found exclusively in symptomatic cases or certain combinations of bacteria are related to symptoms, although the specific roles of each species alone and in combination remain unclear. Nevertheless, one must remember that the virulence of bacteria alone or in combination within a community varies; therefore, bacteria that have been associated with certain symptoms might also be present in asymptomatic infections or even at healthy sites. The overall clinical outcome is a manifestation of synergistic activities and is due to the pathogenicity of an integrated community.

Conclusion The microbial profile of apical lesions differed, with some bacteria tending to occur together and form a unique community. Some bacteria species were associated with symptoms. For example, P. gingivalis fimA type IV was detected only in symptomatic cases. In addition, neither E. faecalis nor C. albicans was found in the persistent apical lesions. Different community profiles can generate similar outcomes, indicating a heterogeneous aetiology related to persistence, not a homogeneous aetiology with a single suspected robust species, as has been alleged previously.

Acknowledgements This study was supported in part by Grants-in Aid for Scientific Research 25463249 (T. T.) and 25293428 (Y. Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The first author is grateful to the Ministry of Education and



Culture, Directorate of Higher Education (DIKTI), Republic of Indonesia for doctoral scholarship and to the Faculty of Medicine, Jenderal Achmad Yani University, Indonesia for supporting doctoral study.

References Amano A, Nakagawa I, Kataoka K, Morisaki I, Hamada S (1999) Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients. Journal of Clinical Microbiology 37, 1426–30. Amano A, Kuboniwa M, Nakagawa I, Akiyama S, Morisaki I, Hamada S (2000) Prevalence of specific genotypes of Porphyromonas gingivalis fimA and periodontal health status. Dental Research 79, 1664–8. Ates M, Akdeniz BG, Sen BH (2005) The effect of calcium chelating or binding agents on Candida albicans. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 100, 626–30. Baca P, Junco P, Arias-Moliz MT, Gonzalez-Rodriguez MP, Ferrer-Luque CM (2011) Residual and antimicrobial activity of final irrigation protocols on Enterococcus faecalis biofilm in dentin. Journal of Endodontics 37, 363–6. Brundin M, Figdor D, Roth C, Davies JK, Sundqvist G, Sjogren U (2010) Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 110, 789–94. Chavez de Paz LE (2007) Redefining the persistent infection in root canals: possible role of biofilm communities. Journal of Endodontics 33, 652–62. Ercan E, Dalli M, D€ ulgergil CT (2006) In vitro assessment of the effectiveness of chlorhexidine gel and calcium hydroxide paste with chlorhexidine against Enterococcus faecalis and Candida albicans. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 102, e27–31. Fujii R, Saito Y, Tokura Y, Nakagawa KI, Okuda K, Ishihara K (2009) Characterization of bacterial flora in persistent apical periodontitis lesions. Oral Microbiology and Immunology 24, 502–5. Gomes BP, Drucker DB, Lilley JD (1994) Associations of specific bacteria with some endodontic signs and symptoms. International Endodontic Journal 27, 291–8. Han YW, Shi W, Huang GT et al. (2000) Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infection and Immunity 68, 3140–6. Handal T, Caugant DA, Olsen I, Sunde PT (2009) Bacterial diversity in persistent periapical lesions on root-filled teeth. Journal of Oral Microbiology 1, 1946. Li H, Takeshita T, Furuta M et al. (2012) Molecular characterization of fungal populations on the tongue dorsum of institutionalized elderly adults. Oral Diseases 18, 771–7.


Love MR (2001) Enterococcus faecalis – a mechanism for its role in endodontic failure. International Endodontic Journal 34, 399–405. Luo G, Mitchell TG (2002) Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR. Journal of Clinical Microbiology 40, 2860–5. Metzger Z, Blasbalg J, Dotan M, Weiss EI (2009) Enhanced attachment of Porphyromonas gingivalis to human fibroblasts mediated by Fusobacterium nucleatum. Journal of Endodontics 35, 82–5. Molander A, Reit C, Dahlen G, Kvist T (1998) Microbiological status of root-filled teeth with apical periodontitis. International Endodontic Journal 31, 1–7. Nagayama M, Sato M, Yamaguchi R, Tokuda C, Takeuchi H (2001) Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis. Letter in Applied Microbiology 33, 122–5. Nair PNR (2004) Pathogenesis of apical periodontitis and the causes of endodontic failures. Critical Reviews in Oral Biology & Medicine 15, 348–81. Nakagawa I, Amano A, Kimura RK, Nakamura T, Kawabata S, Hamada S (2000) Distribution and molecular characterization of Porphyromonas gingivalis carrying a new type of fimA gene. Journal of Clinical Microbiology 38, 1909–14. Noguchi N, Noiri Y, Narimatsu M, Ebisu S (2005) Identification and localization of extraradicular biofilm-forming bacteria associated with refractory endodontic pathogens. Applied and Environmental Microbiology 71, 8738–43. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M (2001) Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. International Endodontic Journal 34, 429–34. Periasamy S, Kolenbrander PE (2009) Aggregatibacter actinomycetemcomitans builds mutualistic biofilm communities with Fusobacterium nucleatum and Veillonella species in saliva. Infecion and Immunity 77, 3542–51. Perry A, Lambert P (2011) Propionibacterium acnes: infection beyond the skin. Expert Review on Anti Infective Therapy 9, 1149–56. Pinheiro ET, Gomes BPFA, Ferraz CCR, Sousa ELR, Teixeira FB, Souza-Filho FJ (2003) Microorganisms from canals of root-filled teeth with periapical lesions. International Endodontic Journal 36, 1–11. Ricucci D, Siqueira JF Jr (2008) Apical actinomycosis as a continuum of intraradicular and extraradicular infection: case report and critical review on its involvement with treatment failure. Journal of Endodontics 34, 1124–9. R^ ocas IN, Siqueira JF Jr (2011) Comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study. Journal of Endodontics 37, 143–50. Saber MH, Schwarzberg K, Alonaizan FA et al. (2012) Bacterial flora of dental periradicular lesions analyzed by the 454-pyrosequencing technology. Journal of Endodontics 38, 1484–8.


11


Saito A, Inagaki S, Kimizuka R et al. (2008) Fusobacterium nucleatum enhances invasion of human gingival epithelial and aortic endothelial cells by Porphyromonas gingivalis. FEMS Immunology and Medical Microbiology 54, 349–55. Sassone LM, Fidel RA, Faveri M et al. (2008) A microbiological profile of symptomatic teeth with primary endodontic infections. Journal of Endodontics 34, 541–5. Siqueira JF Jr, R^ ocas IN (2003) Polymerase chain reaction detection of Propionibacterium propionicum and Actinomyces radicidentis in primary and persistent endodontic infections. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 96, 215–22. Siqueira JF, R^ ocas IN (2004) Polymerase chain reaction– based analysis of microorganisms associated with failed endodontic treatment. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 97, 85–94. Siqueira JF Jr, R^ ocas IN (2008) Clinical implications and microbiology of bacterial persistence after treatment procedures. Journal of Endodontics 34, 1291–301. Soejima T, Iida K, Qin T, Taniai H, Seki M, Yoshida S (2008) Method to detect only live bacteria during PCR amplification. Journal of Clinical Microbiology 46, 2305–13. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB (2006) Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of Endodontics 32, 93–8. Su L, Gao Y, Yu C, Wang H, Yu Q (2010) Surgical endodontic treatment of refractory periapical periodontitis with extraradicular biofilm. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 110, e40–4. Sunde PT, Olsen I, Debelian GJ, Tronstad L (2002) Microbiota of periapical lesions refractory to endodontic therapy. Journal of Endodontics 28, 304–10. Sundqvist G, Figdor D, Persson S, Sj€ogren U (1998) Microbiologic analysis of teeth with failed endodontic treatment

12


and the outcome of conservative re-treatment. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 85, 86–93. Takeshita T, Nakano Y, Yamashita Y (2007) Improved accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standard definition. Oral Microbiology and Immunology 22, 419–28. Takeshita T, Nakano Y, Kumagai T (2009) The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status. The ISME Journal 3, 65–78. Tong Z, Ling J, Lin Z, Li X, Mu Y (2013) The effect of MTADN on 10 Enterococcus faecalis isolates and biofilm: an in vitro study. Journal of Endodontics 39, 674–8. Waltimo TM, Siren EK, Torkko HL, Olsen I, Haapasalo MP (1997) Fungi in therapy-resistant apical periodontitis. International Endodontic Journal 30, 96–101. Waltimo TMT, Sen BH, Meurman JH, Orstavik D, Haapasalo MPP (2003) Yeasts in apical periodontitis. Critical Reviews in Oral Biology & Medicine 14, 128–37. Wang J, Jiang Y, Chen W, Zhu C, Liang J (2012) Bacterial flora and extraradicular biofilm associated with the apical segment of teeth with post-treatment apical periodontitis. Journal of Endodontics 38, 954–9. Zhu X, Wang Q, Zhang C, Cheung GS, Shen Y (2010) Prevalence, phenotype, and genotype of Enterococcus faecalis isolated from saliva and root canals in patients with persistent apical periodontitis. Journal of Endodontics 36, 1950–5. Zoletti GO, Siqueira JF Jr, Santos KR (2006) Identification of Enterococcus faecalis in root-filled teeth with or without periradicular lesions by culture-dependent and-independent approaches. Journal of Endodontics 32, 722–6.


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