Prevalence analysis of putative periodontal pathogens in patients with aggressive periodontitis and healthy elderly. A molecular studyLilian Edesi-Neuss111.121.234561.2.11.2.271.2.381.2.491.2.51.2.6101.3111.4121.4.11.4.21.4.3131.4.41.51.61.6.11415161.6.21.71721733.117183.21920213.33.4222324253.5263.6273.73.7.1283.7.2293.83043055.130315.2325.3604#29933615#30834626#286614#27635614#30736605#6905.4376376.13839406.26.2.141426.2.2436.2.36.2.4446.2.56.2.6456.2.76.2.86.2.9466.2.106.2.1147486.36.4495075085099.1519.29.2.19.2.1.15253549.2.1.29.2.1.39.2.1.455569.2.1.59.2.1.657589.2.29.2.2.1599.2.2.29.2.2.39.2.2.4609.2.2.59.2.2.69.2.2.7619.2.2.89.2.2.99.2.2.10629.2.2.119.2.39.2.3.19.2.3.263References Danksagung Lebenslauf Eidestattliche ErklärungenTable of contentsHelp Supplement
Tables

1) Actinobacillus actinomycetemcomitans

ATCC 43718

DSM

sequencing

2) Actinobacillus actinomycetemcomitans

ATCC 33384

Conrads

sequencing

3) Actinobacillus actinomycetemcomitans

serotyp a

Mombelli

--

4) Leptotrichia buccalis

MCCM 00448

Mutters

sequencing

5) Pasteurella haemolytica

ATCC 33396

Mutters

sequencing

6) Haemophilus influenzae

ATCC 33391

Mutters

sequencing

7) Haemophilus influenzae

klin. Isolat

Conrads

--

8) Haemophilus aphrophilus

NCTC 55906

Gmür

sequencing

9) Haemophilus paraphrophilus

ATCC 29241

Mutters

sequencing

10) Porphyromonas gingivalis

ATCC 33277

DSM

sequencing

11) Prevotella intermedia

ATCC 25611

DSM

sequencing

12) Porphyromonas asaccharolytica

ATCC 25260

Gmür

sequencing

13) Prevotella nigrescens

NCTC 9336

Gmür

sequencing

14) Prevotella oralis

MCCM 00684

Mutters

--

15) Prevotella buccalis

ATCC 33690

Mutters

API

16) Capnocytophaga ochracea

ATCC 27872

Gmür

sequencing

17) Capnocytophaga sputigena

ATCC 33612

Gmür

sequencing

18) Capnocytophaga gingivalis

ATCC 33624

Conrads

sequencing

19) Campylobacter rectus

ATCC 33238

Mombelli

sequencing

20) Campylobacter concisus

ATCC 33236

Gmür

sequencing

21) Tannerella forsythensis

ATCC 43037

Olson

sequencing

22) Bacteroides gracilis

ATCC 33236

Gmür

sequencing

23) Bacteroides fragilis

ATCC 25285

Charité

--

24) Eikenella corrodens

CCUG 2138

Gmür

sequencing

25) Kingella kingae

ATCC 23330

DSM

sequencing

26) Veillonella parvula

ATCC 10790

Gmür

sequencing

27) Veillonella dispar

ATCC 17748

DSM

--

28) Klebsiella pneumoniae

ATCC 23357

Charité

--

29) Fusobacterium nucleatum

ATCC 25586

Mombelli

sequencing

30) Fusobacterium odoratum

MCCM 02932

Mutters

sequencing

31) Neisseria lactamica

ATCC 23970

Charité

--

32) Streptococcus mutans

ATCC 35668

Virchow

--

33) Streptococcus intermedius

ATCC 27335

Virchow

--

34) Actinomyces pyogenes

ATCC 19411

Virchow

--

35) Actinomyces israelii

ATCC 10048

Virchow

--

36) Actinomyces naeslundii

ATCC 12104

Virchow

--

37) Actinomyces viscosus

ATCC 15987

Virchow

--

38) Eubacterium lentum

ATCC 25559

Mutters

--

39) Selenomonas sp.

Clin. strain

Mutters

--

40) Fusobacterium simiae

CCUG 16798

CCUG

sequencing

41) Fusobacterium periodonticum

CCUG 14345

CCUG

--

42) Fusobacterium necrophorum

NCTC 25286

Mombelli

sequencing
Table 1. Strain designations, original sources, control identification method used in the study.

1) Actinobacillus actinomycetemcomitans

ATCC 43718

fac. anaerob.

TSBV

2) Actinobacillus actinomycetemcomitans

ATCC 33384

fac. anaerob.

TSBV

3) Actinobacillus actinomycetemcomitans

serotyp a

fac. anaerob.

TSBV

4) Leptotrichia buccalis

MCCM 00448

anaerobic

Fluid thyogl.

5) Pasteurella haemolytica

ATCC 33396

aerobic

Col. agar

6) Haemophilus influenzae

ATCC 33391

microaerophil.

anaer. Col. agar

7) Haemophilus influenzae

Clinical strain

microaerophil.

anaer. Col. agar

8) Haemophilus aphrophilus

NCTC 55906

microaerophil.

anaer. Col. agar

9) Haemophilus paraphrophilus

ATCC 29241

microaerophil.

anaer. Col. agar

10) Porphyromonas gingivalis

ATCC 33277

anaerobic

anaer. Col. agar

11) Prevotella intermedia

ATCC 25611

anaerobic

anaer. Col. agar

12) Porphyromonas asaccharolytica

ATCC 25260

anaerobic

anaer. Col. agar

13) Prevotella nigrescens

NCTC 9336

anaerobic

anaer. Col. agar

14) Prevotella oralis

MCCM 00684

anaerobic

anaer. Col. agar

15) Prevotella buccalis

ATCC 33690

anaerobic

anaer. Col. agar

16) Capnocytophaga ochracea

ATCC 27872

anaerobic

anaer. Col. agar

17) Capnocytophaga sputigena

ATCC 33612

anaerobic

anaer. Col. agar

18) Capnocytophaga gingivalis

ATCC 33624

anaerobic

anaer. Col. agar

19) Campylobacter rectus

ATCC 33238

anaerobic

FUM+supplem.

20) Campylobacter concisus

ATCC 33236

anaerobic

FUM +supplem.

21) Bacteroides gracilis

ATCC 33236

anaerobic

anaer. Col. agar

22) Bacteroides fragilis

ATCC 25285

anaerobic

anaer. Col. agar

23) Eikenella corrodens

CCUG 2138

anaerobic

Col. Agar

24) Kingella kingae

ATCC 23330

aerobic

Col. agar

25) Veillonella parvula

ATCC 10790

anaerobic

FUM +supplem.

26) Veillonella dispar

ATCC 17748

anaerobic

anaer. Col. agar

27) Klebsiella pneumoniae

ATCC 23357

aerobic

Col. Agar

28) Fusobacterium nucleatum

ATCC 25586

anaerobic

ETSA

29) Fusobacterium odoratum

MCCM 02932

anaerobic

anaer. Col. agar

30) Neisseria lactamica

ATCC 23970

microaerophil.

Col. Agar

31) Streptococcus mutans

ATCC 35668

aerobic

Col. Agar

32) Streptococcus intermedius

ATCC 27335

aerobic

Col. Agar

33) Actinomyces pyogenes

ATCC 19411

anaerobic

anaer. Col. agar

34) Actinomyces israelii

ATCC 10048

anaerobic

anaer. Col. agar

35) Actinomyces naeslundii

ATCC 12104

anaerobic

anaer. Col. agar

36) Actinomyces viscosus

ATCC 15987

anaerobic

anaer. Col. agar

37) Eubacterium lentum

ATCC 25559

anaerobic

anaer. Col. agar

38) Selenomonas sp.

anaerobic

anaer. Col. agar

39) Fusobacterium simiae

CCUG 16798

anaerobic

ETSA

40) Fusobacterium periodonticum

CCUG 14345

anaerobic

ETSA

41) Fusobacterium necrophorum

NCTC 25286

anaerobic

ETSA
Table 2. The bacterial species, respective culture media and growth conditions.

List of used mediums and chemicals Mediums FUM - Fluid Universal Medium

(R. Gmür, B. Guggenheim, Infect. Immun. 1983; 42: 459-470)

Stock-solutions (stored at 4°C)

Hemin:

0.1 M KOH
Ethanol conc.
Dist. Water
Hemin-chlorid

100ml
50ml
50ml
200mg

Menadione:

Menadione
Ethanol conc.

25mg
50ml

Reduced transport fluid

RTF 1:

K2HPO4dist. water ad

6g
1000ml

RTF 2:

NaCl
(NH4)2SO4
KH2PO4
MgSO4 x 7 H2O
distilled water ad

12g
12g
6g
2.5g
1000ml

Solution A:

Solution B:

Bacto Tryptone
Yeast extract
KNO3
NaCl
Hemin solution 1 ml

10g
5g
1g
2g
1ml

Glycose
Cysteine-HCl
Na2CO3
Dist. Water
Filter-sterilized

3g
0.5g
0.5g
50ml

Autoclaved 20 min. at 120°C and stored at 4°C

Production of FUM:

RTF 1

75 ml

RTF 2

75 ml

Menadione solution

2 ml

Filter-sterilized. Pooled into Solution B and added to Solution A.
5 % heat inactivated (30 min at 56°C) horse serum (GIBCO) was added.

Additional components:

1% N-Acetylmuramic acid C11H19NO8 10mg in
1 ml dist. water

For C. concisus, C. rectus :

Fumaric acid disodiumsaline C4H2Na2O4 1 g +
Sodiumformiat CHNaO2 1 g in 10 ml dist. water

For V. parvula :

Putrescin C4H12N2 4,5 mg in 10 ml dist. water

Columbia agar

40 mg/l

Columbia agar (Becton-Dickenson)

5 mg/l

Hemin

50 µg/l

Vitamin K

5%

Sheep blood

TSBV - Trypticase-Soy-Bacitracin-Agar (J. Slots, J Clin Microbiol 1982: 15: 606)

40g/l

Trypticase soy agar

75 mg/l

Bacitracin

5 mg/l

Vancomycin

1 g/l

Yeast extract

2g/l

Glycose

10%

Horse serum

ETSA - Enriched-Trypticase-Soy-Agar

Bulk preparation A

20 ml
4 ml
4 ml
Distilled water ad
4 g
2 g
2 g
Autoclaved, cooled, freezed

10% Potassium nitrate
Sodium lactate syrup
Hemin
2000 ml
Yeast extract
Sodium succinate
Sodium formate

Bulk preparation B

25 g
19 g
Dist. water ad 500 ml
Autoclaved, cooled in 50°C water bath, added to prep. A

Trypticase soy broth
Agar

Filter sterilized ingredients added:
25 ml
2 ml
2 ml
0.4 g
0.1 g
1 g
0.4 g
30 ml


Dist. water
Menadione
Fumarate
Cysteine
Dithiothreitol
Glycose
Sodium carbonate
Sheep blood

Trypticase Soy Yeast Extract Medium

30 g
3.0 g
15.0 g
Distilled water ad 1000 ml
pH adjusted to 7.0 - 7.2

Trypticase soy broth
Yeast extract
Agar

RTF

A) Standard medium 1

K2HPO4 autoclaved

B) Standard medium 2

12 g/l
12 g/l
6 g/l
2.5 g/l
Autoclaved

NaCl
(NH4)2SO4
KH2PO4
MgSO4

C)

38 g/l

0.1M EDTA

D)

8 g/100 ml

Na2CO3

E)

0.2 g/20 ml

1% Dithiothreitol (DTT)

F)

0.1 g/100 ml

Resazurin

Preparation:

75 ml
75 ml
10 ml
5 ml
20 ml
1 ml
25% Glycose
Distilled water ad 1000 ml, filter-sterilize

Standard medium 1
Standard medium 2
Portion C
Portion D
Portion E
Portion F

Buffers Maleic acid buffer

0.1 M Maleic acid
0.15 M NaCl
Adjust pH 7.5 with solid NaOH

 1% Blocking solution

Blocking reagent 1g
Maleic acid buffer 100 ml
Dilute by stirring at 65°C, autoclave, store at 4°C

 Detection buffer

0.1 M Tris-HCl
0.1 M NaCl
Adjust pH 9.5 with solid NaOH

 Hybridization buffer

5 x SSC
1% Blocking solution
0.1% N-lauroylsarcosine
0.02% Sodium dodecyl sulfate (SDS)

 Washing buffer 0:

5 x SSC
0.2% SDS

 Washing buffer 1:

2 x SSC
0.1% SDS

 Washing buffer 2:

0.1x SSC
0.1% SDS

 Stripping solution

0.2 M NaOH
0.1% SDS

 Phosphate buffered saline (PBS)

NaCl 8 g
KCl 0.2 g
Na2HPO4 1.44 g
KH2PO4 0.24 g
ad 1 l distilled water, adjust pH 7.4

 Lysis buffer

500 mM Tris-HCl pH 9.0
20 mM EDTA
10 mM NaCl
1% SDS

 Stop-Solution

9,5 ml Formamid
0.4 ml EDTA (500 mM)
5.0 mg Bromphenolblau
5.0 mg Xylencyanol FF
0.1 ml Dist. water

 Gels 1.2% Agarose gel

1.2 mg Agarose
100 ml TBE buffer
1µl Ethidiumbromid

 Polyacrylamidegel

21 g Urease
6 ml 10xTBE buffer
4.5 ml Long-Ranger (40%)
Dist. water ad 50 ml
Stirred at 50°C, filter-sterilized
240 µl APS (10%)
24 µl TEMED