DNA Terminal-Restriction Fragment Length Polymorphism (T-RFLP)

T-RFLPT-RFLP

In this study, oral microflora was examined in relationship toperiodontitis and malodor, and was further analyzed with febrile days and pneumonia in institutionalized elderly. To characterize the composition of oral microflora, chromosomal DNA was extracted from bacteria in saliva or tongue coat and Terminal-Restriction Fragment Length Polymorphism analysis was carried out using the extracted bacterial DNA as template. The results indicated that T-RFLP pattern of salivary bacteria was closely associated with the prevalence of periodontitis and malodor. In addition, T-RFLP pattern in tongue coat was also associated with febrile days and pneumonia in institutionalized elderly.

T-RFLP

Study of effect of oral microflora on oral and general health

1

九州大学・大学院歯学研究院・教授

1

16S rRNAPCR

Terminal-Restriction Fragment Polymorphism (T-RFLP)

16S rRNA

PCR

T-RFLPT-RFLP

T-RFLP

T-RFLP

T-RFLP

(1)

DNA

6-FMA 8F (5'-AGAGTTTGATYMTGG CTCAG-3')

HEX 806R(5'-GGACTACCR GGGTATCTAA-3')

PCR 16S rRNAHaeIII

T-RFLP T-RFLP

(2)

15 40 200102 98 28.1

T-RFLPI, II, III

(3)

12 74 240102 98 28.1

T-RFLPI, II, III, IV

4

(4)

65

343 85 258 85.8

T-RFLPA, B, C, D

4

37.5

(5)

3400

2612

T-RFLPI, II, III

(1)

III

I II I

FAM19, FAM33, HEX16, HEX22 II

FAM47, HEX32 IIIFAM17. FAM65, HEX12, HEX24

TRFMAW

III

III,

(2)

I

(VSC) VSC

IIIV

Cluster I Cluster II Cluster III (n = 41) (n = 74) (n = 85)

( ) ( SD) 28.9 5.6 28.8 6.6 27.1 6.2 NS b

(%) 14 (34.1) 41 (55.4) 43 (50.5) NS c

(%) 13 (31.7) 16 (21.6) 12 (14.1) NS c

( SD) 0.2 0.6 0.4 1.0 0.2 0.7 NS b

DMFT 9.4 6.4 10.1 7.1 7.8 5.4 NS b

( SD) 27.6 18.1 18.9 19.8 14.6 13.5 I > II, I > III b

( > 4 mm) 10.1 15.4 9.0 15.1 3.7 6.4 I > III, II > III b

10 (24.3) 36 (48.6) 55 (64.7) P < 0.001 c >20% 9 (21.9) 14 (18.9) 2 (2.3) P < 0.001 c

(x 108 deduced CFU per ml) 6.7 7.9 2.6 2.7 1.5 2.0 I > II, I > III ba P values < 0.05 . NS: .

c Fisher's exact test. P values < 0.05 .

a

b Steel-Dwass test. P values < 0.05 .

28.9 5.6

14 (34.1)

13 (31.7)

0.2 0.69.4 6.4

27.6 18.110.1 15.410 (24.3)9 (21.9)6.7 7.9

28.8 6.6

41 (55.4)

16 (21.6)

0.4 1.010.1 7.1

18.9 19.89.0 15.136 (48.6)14 (18.9)2.6 2.7

27.1 6.2

43 (50.5)

12 (14.1)

0.2 0.77.8 5.4

14.6 13.53.7 6.455 (64.7)2 (2.3)

1.5 2.0

T-RFLP

Principle component 1 (40.0%) 0 0.5

Cluster I Cluster II

Cluster III

- 0.5

Cluster I

Cluster II

#17FAM

#24HEX

#65FAM

#32HEX

#33FAM #19FAM

#22HEX

#8HEX

#16HEX

#12HEX #27FAM

#47FAM

Cluster III

VSC (ppm)

(n = 41) (n = 64) (n = 71) (n = 64) (95% CI) (n (%))

T-RFLP Cluster I 21 (51.2) 13 (20.3) 9 (12.7) 0 (0) 1 Cluster II 6 (14.6) 19 (29.7) 33 (46.5) 32 (50.0) 11.1 (5.4-23.2)*** Cluster III 10 (24.4) 22 (34.4) 14 (19.7) 16 (25.0) 5.0 (2.4-10.5)*** Cluster IV 4 (9.8) 10 (15.6) 15 (21.1) 16 (25.0) 10.2 (4.5-23.3)***

*P<0.05, **P<0.01, ***P<0.001.

VSC

II

IV

(3)

H2S CH3SH Cluster I 1 1 Cluster II 10.6 (5.0-22.3)*** 14.6 (6.9-30.8)*** Cluster III 5.9 (2.7-12.7)*** 5.5 (2.5-11.7)*** Cluster IV 16.5 (7.1-38.7)*** 8.4 (3.7-19.0)***

5mm 1 4.9mm 0 0.9mm

N=343

9 = 0; (%) Hazard (%) Odds (95% CI) (95% CI) 1.0 (1.0-1.1) 1.0 (1.0-1.1)

8 (10.7) 1 7 (11.5) 1

27 (10.1) 0.9 (0.4-2.4) 39 (16.4) 0.7 (0.2-2.4)

A 3 (3.0) 1 3 (3.4) 1 B 8 (22.2) 2.7 (0.6-12.6) 15 (11.1) 4.4 (1.1-17.8)* C 13 (8.5) 4.0 (1.1-15.1)* 13 (52.0) 10.5 (2.0-55.5)** D 11 (20.4) 4.9 (1.2-21.1)* 15 (29.4) 11.6 (2.3-57.8)**

or 11 (7.5) 1 17 (12.9) 1 or 24 (12.2) 3.2 (1.4-7.3)** 29 (17.4) 3.7 (1.4-9.6)**

(mm)

1 4.9 14 (10.2) 2.9 (1.1-8.0)* 18 (14.9) 2.7 (1.0-7.3)* 0.9 14 (26.9) 7.9(2.8-22.4)** 16 (39.0) 10.3 (2.9-36.4)**

F29

F68

F53

F32

F56

F90

R29

R20 R13

R30

R39 R10 R43

R12 F36

F42 F57 F80

: Cluster I : Cluster II : Cluster III : Cluster IV

Principal component 1 (45.9%) 0 0.4 -0.4

F29

F56

F90

R30

R12

F42

F32

R20R13

RF80

I II

IV

III Streptococcus Granulicatella Rothia Treponema

Prevotella Veillonella

Fusobacterium Parvimonas Porphyromonas Lautropia Neisseria Haemophilus Aggregatibacter

T-RFLP

Cluster A

Cluster C: Cluster D:

Veillonella

Prevotella Treponema

Streptococcus

Rothia

9 = 0;

(%) Hazard (%) Odds (95% CI) (95% CI)

28 (13.1) 1 37 (20.2) 1

7 (5.4) 1.0 (0.4-2.8) 9 (7.8) 1.5 (0.5-4.0)

3 (3.0) 1 2 (2.3) 1 10 (9.0) 2.6 (0.7-10.1) 9 (8.9) 4.6 (0.8-25.3)

22 (16.8) 1.3 (0.3-6.4) 35 (31.5) 6.7 (1.1-39.4)*

9 (4.8) 1 9 (5.6) 1 9 (8.0) 1.4 (0.5-3.9) 14 (13.5) 1.5 (0.5-4.1) 17 (38.6) 7.0(1.7-28.9)** 23 (67.6) 9.5 (2.1-42.9)**

( ) 0 23 (7.9) 1 33 (12.9) 1 1 - 4 5 (17.2) 2.7 (0.9-8.2) 7 (28.0) 4.0 (1.1-15.1)*

2.8 (1.0-7.5)* 6 (33.3) 3.6 (0.8-15.6)

C, D

Prevotella Treponema Veillonella

Streptococcus Rothia

(4)

I

II III

Neisseria HaemophilusI

II,IIIPrevotella

Veillonella StreptococusRothia

( 1. Takeshita T, Nakano Y, Yamashita Y, Improveded accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standarddefinition. Oral MicrobiolImmunol 22(6): 419 428, 2007. 2. Nakano Y, Takeshita T, Kamio N, Shiota S, Shibata Y, Yasui M, Yamashita Y. Development and application of a T-RFLP data analysis method using correlation coefficient matrices. J Microbiol Methods 75(3):501-505, 2008. 3. Takeshita T, Nakano Y, Kumagai T, Yasui M, Kamio N, Shibata Y, Shiota S, Yamashita Y. The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status. ISME J 3(1):65-78, 2009. 4. Takeshita T, Suzuki N, Nakano Y, Shimazaki Y, Yoneda M, Hirofuji T, Yamashita Y. Relationship between oral malordor and the global composition of indigenous bacterial populations in salaiva. Appl Environ Microbiol. in press 2010. 5. Takeshita T,Tomioka M, Shimazaki Y, Matsuyama M, Koyano K, Matsuda K, Yamashita Y. J Am Geriatr Soc, in press, 2010.

15

292007 7 8 2.

T-RFLP30 / 80

2007 12 13

3.

812008 3 24

. Takeshita1 T, Suzuki N, Nakano Y, YonedaM, Shibata Y, Shiota S, Kamio N, Hirofuji T, Yamashita Y: Correlation between oral malodor and bacterial composition in saliva, 86th General Session & Exhibition of the IADR, 2008 7 3

, Toronto, Canada. 5.

302008 7 5

6.

502008 9 23

Yasui M, Takeshita T, Nakano Y, Shimazaki

Y, Shibata Y, Shiota S, AkifusaS, Yamashita Y:The change of microbial populations during plaque development, 8th Congress of Asian Academy of Preventive Dentistry, 2008 11 6

, Cheju, Korea. 8. Tomioka M, Shimazaki Y, Takeshita T, MatsuyamaM, Koyano K,Yamashita Y: Relationship between the tongue conditions and pyrexia in the elderly8th Congress of Asian Academy of Preventive Dentistry, 2008 11 6

, Cheju, Korea. 9.

25

2009 1 10.

312009 7 5

11.Tomioka M, Takeshita T, Shimazaki Y, Matsuyama M, Koyano K, Yamashita Y: Comprehensive analysis of the bacterial flora of tongue coating and pneumonia risk in the institutionalized elderly, First European Congress on Microbial Biofilms,2009 9 5 , Rome, Italia, 12.

582009 10 9

13. Takeshita T, Suzuki N, Nakano Y, Shimazakia Y, Yoneda M, Hirofuji T, Yamashita Y: Relationship between the global composition of indigenous bacterial populations in saliva and oral malodor, International symposium on "oral health science" in 2010, 2010 2 6 , Fukuoka, Japan. 14.

832010 3 27 15.

832010 3 28

21.

p87-103, MI

2009.

2. Yamashita Y, Takeshita T: A comprehensive evaluation of the virulence of oral flora, p251-267, in "Informatics in Oral Medicine", ed: Daskalaki A, Medical Information Science Reference, New York, USA.

2010-064709 2010-03-19