Evaluation of the diversity of two species of the genus Propionibacterium : Mass

Spectrometry versus Triple-Locus Sequence Analysis

F. Valence1, P. Bouvet2, J. Jardin3, M. Dalmasso3, L. Motreff4, C. Pissis4, S. Lortal3, C. Bizet4

and D. Clermont4

1: CIRM-BIA, INRA, UMR 1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France

2: Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, Paris, France

3: INRA, UMR 1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France

4: Collection de l'Institut Pasteur, F-75724 Paris Cedex 15, France

SEVENTH FRAMEWORK PROGRAMME

THEME: INFRA-2008-1.1.2.9

Biological Resources Centres (BRCs) for micro-organisms

GRANT agreement N° 228310

2

Genus Propionibacterium:

phenotypic characteristics

Gram-positive, rod-shaped bacteria that may bifurcate or even

branch, nonspore-forming, anaerobic (microaerophilic)

Generally catalase positive

Characterized by the large amounts of propionic acid during

growth

3

Propionibacterium freudenreichii: mainly isolated from dairy sources

important function in the cheese industry involved in the

hole and flavor formations in Swiss-type cheese

probiotic potential: production of vitamin B12 and inhibition

of the unwanted microflora

Propionibacterium acnes: isolated from human (commensal of the skin, mouth…)

opportunistic pathogen (acne, endocarditis, prosthetic joint

infections…)

Propionibacterium freudenreichii

and Propionibacterium acnes:Habitat and Functions

4

Aims of the study

To determine the degree of molecular diversity within

P. freudenreichii and P.acnes at species and clonal level

To compare the ranking of strains according to the methodology applied : triple locus sequencing or mass spectrometry

To evaluate the reproducibility of the mass spectrometry method (two equipments, two laboratories and two data analysis methods)

5

Materials

20 strains of P. freudenreichii

from CIRM-BIA, INRA selected in

order to offer a large diversity in

terms of biotope (geographic and

habitat) 2 subspecies (freudenreichii and

shermanii described on the basis of

lactose fermentation and nitrate

reductase activity)

21 strains of P. acnes from Institut Pasteur isolated from human between 1920-1960 and 2000-2009

3 phylogenetic groups described by sequence analysis of recA and tlygenes: Types I, II, III

Origin P. freudenreichii

subsp.

freudenreichii

P. freudenreichii

subsp.

shermanii

raw milk / 6

cheese 1 8

"vegetal"

(hay, straw,

wheat)

/ 4

unknown / 1

Origin P. acnes

blood 7

Various (acne,

abscess..)

9

unknown 5

6

Methods

Gene sequencing: P. acnes: 16S rRNA, rpoB, adk, gyrB

P. freudenreichii: 16S rRNA, rpoB, adk, fumC

MALDI-TOF Mass Spectrometry Based on mass analysis of protein

composition of the bacterial cells

Generation of spectra

Bruker Daltonics autoflex (IP):

Comparison with high quality reference

spectra, generated by culture collections

Applied Biosystems equipment

(INRA/CIRM):

Statistical analysis of the data:

Principal component analysis

(Wahl et al. 1999)

7

Results: Phylogenetic tree based on 16S rRNA gene sequences for P. acnes

- A. turencis was used as outgroup. Neighbourg-joining method.

- Numbers represent bootstrap values of 100 replicates

P.Acnes type I108-02228-08391-00139-0953-117T P.acnes type I219-08A17954.15860.42217-0253.119266-02483-03P.Acnes type IP.Acnes type IIIP.Acnes type IIIP.Acnes type IIIP.Acnes type IIIP.Acnes type III81-02P.Acnes type II27-08335-02110-06P.Acnes type IIP.Acnes type II329-00511-0071-01114-09

103261T Propionibacterium avidum105357T Actinomyces turicensis

55

62

60

99

0.01

Type I

Type III

Type II

- The 3 types previously

described are found among the

strains studied.

- The place of strain 71-01 is not

well defined by 16S rRNA gene.

- 114-09 is distant from other

strains. Result was confirmed by

using the genes rpoB, gyrB and

adk.

Control strains of the 3

types indicated in bold

8

Phylogenetic tree based on combinated adk, rpoB

and gyrB gene sequences for P. acnes

- The 3 types are well defined

- Type I contains 2 groups not seen

with 16S rRNA and not already

described.

CIP 60-42391-00

266-02CIP 53-119217-02CIP 53-117T483-03

CIP 54-15871-01108-02139-09

228-08CIP A179

81-0227-08

110-06335-02219-08329-00

63

35

52

100

15

100

88

99

0.002

- No relation between strain

origins and clusters.

Colour Code : blood, various,

unknown

- All the old strains belong to Type I

- No Type specificity for the strains

recently isolated.

Type I

Type II

Type III

9

Phylogenetic trees based on combinated adk, rpoB

and fumC gene sequences for P. freudenreichii

DNA-DNA homology within the P. freudenreichii

species is very high (more than 86%)

- Triple locus sequence analysis does not

permit to separate the 2 subspecies.

CIRM-BIA683CIRM-BIA456

CIRM-BIA514TL600CIRM-BIA141

CIRM-BIA516CIRM-BIA515CIRM-BIA686

CIRM-BIA527CIRM-BIA121 P. freudenreichii subsp. freudenreichii

CIRM-BIA693CIRM-BIA7

CIRM-BIA513CIRM-BIA508CIRM-BIA1T P. freudenreichii subsp. shermaniiCIRM-BIA688

CIRM-BIA689CIRM-BIA692CIRM-BIA8CIRM-BIA512

99

7763

67

0.0005

- No relation between strain origin and

clusters.

Colour Code : Cheese, raw milk, wheat

Results confirmed by MLST study of 7

genes on 100 strains.

See Area 2 - Poster Number: 17

"Lineages with broad dairy biotope

ranges and phenotypic variability in

Propionibacterium freudenreichii

revealed by multilocus sequence

typing"

10

+TOF MS: 60 MCA scans from BIA121_25.wiffa=3.57326353521455530e-004, t0=-3.37680250247140070e+001

Max. 138.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

138

Inte

nsity

, cou

nts

5650.2760

8418.6259

9545.0821

7859.6347 8222.8489

6063.425310211.8612

6532.6797

7042.8393

4421.3684

6888.05612600.5916 3866.2730 5183.03508988.0516 10878.3449

10088.85486953.823710439.46456502.4825 7816.29185139.1079 8829.18766700.7972

7658.42766808.9025 10406.27775686.1622 8046.4845 13534.2838

BIA_121

+TOF MS: 60 MCA scans from BIA141_25.wiffa=3.57326353521455530e-004, t0=-3.37680250247140070e+001

Max. 114.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

114

Inte

nsity

, cou

nts

7239.8068

7012.8622

3867.3105

9545.5076

8222.7988

6812.34458419.6725

7860.4448

6041.54637356.3042

5651.0369

10150.77436533.5592

6888.0482

4423.41468422.5274

10877.33497245.55876021.40755185.1722

8988.04497842.5275

6709.8038 9504.30402600.6004

6417.92645139.9518

7095.6173 8968.18409598.54887416.7621

2537.46845038.0748 6576.7373

3296.86108032.2679 9336.6367 10073.43887552.0599

2697.599513808.7165 14914.4271

4683.71714057.2767

BIA_141

+TOF MS: 60 MCA scans from BIA514_50.wiffa=3.57326353521455530e-004, t0=-3.37680250247140070e+001

Max. 131.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

Inte

nsity

, cou

nts

7012.9668

7241.1422

8419.7723

9546.3601

3867.4143

6064.5849 7043.9160 8224.5297

5648.0778

6532.68834420.5742 10212.6103

6888.2029 7861.490310878.62935183.0641

6812.7703

6514.36238989.0085 10133.72252600.5970 7279.7986

8204.0703 9339.20715086.8907

7746.82156101.5899

7526.9201 9180.00975051.7851 14932.71499565.04762402.6626 10425.22044458.3251 6185.7424

BIA_514

+TOF MS: 60 MCA scans from P228-08_50.wiffa=3.57327121350785870e-004, t0=-3.36406827176178920e+001

Max. 376.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

376

Inte

nsity

, cou

nts

7180.0331

7001.4589

9589.5122

2601.83485168.81654371.3740

P228-08

+TOF MS: 60 MCA scans from P219-08_25.wiffa=3.57327121350785870e-004, t0=-3.36406827176178920e+001

Max. 358.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

358

Inte

nsity

, cou

nts

7179.5818

6986.4498

9589.4848

4372.88622601.5938 5168.9323 6806.8667 7256.5195

+TOF MS: 60 MCA scans from P27-08_75.wiffa=3.57327121350785870e-004, t0=-3.36406827176178920e+001

Max. 226.0 counts.

2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4m/z, amu

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

226

Inte

nsity

, cou

nts

7251.2177

9589.2980

2600.73839547.2606

4373.47476985.07875170.7827

6785.6615

6576.32083942.0568 8667.1882

P27-08

P219-08

P. a

cnes

P. f

reu

den

reic

hii

MALDI-TOF results: examples of profiles

2 types of profiles in relation with the species of Propionibacterium

One species = one specific profile

11

MALDI-TOF results:

Principal Component Analysis

2 groups corresponding to the 2 species P. freudenreichii and

P. acnes

12 spectra

obtained for

each strain

12

MALDI-TOF results:

Principal Component Analysis: strain 114-09

Strain 114-09 : away from other strains according to phylogenetic trees based on 16S

rRNA and genes rpoB, gyrB and adk, it also shows an atypical mass profil

12 spectra

obtained for

each strain

13

MALDI-TOF results:

Comparison between the two equipments used

- Similar results obtained whathever the equipment and analyse type performed

- Regarding P. acnes, the groups obtained obviously reflect the molecular types

Bruker Daltonics autoflex (IP)

Type I

Type II and III

P. freudenreichii

P. acnes

01002003004005006007008009001000

689 P. freudenreichiiTL600 P.freudenreichii693 P. freudenreichii514 P. freudenreichii527 P. freudenreichii7 P. freudenreichii512 P. freudenreichii515 P. freudenreichii8 P. freudenreichii683 P. freudenreichii686 P. freudenreichii688 P. freudenreichii508 P. freudenreichii513 P._freudenreichii516 P. freudenreichii1 P. freudenreichii108.02 P.acnes71.01_P. acnes229.08 P. acnes139.09 P. acnesCIP A179 P. acnes219.08 P. acnes228.08 P. acnes483.03 P. acnes266.02 P. acnesCIP 54.158 P. acnesCIP 60.42 P. acnesCIP 53.117 T P. acnesCIP 53.119 P. acnes110.06 P. acnes27.08 P. acnes81.02 P. acnes335.02 P. acnes511.00 P. acnes114.09 P. acnes

MSP Dendrogram

Distance Level

P. acnes

P. freudenreichii

Applied Biosystems

equipment (INRA/CIRM)

14

Conclusions (1)

Congruence between individual housekeeping gene trees.

No phylogenetic resolution of the two P. freudenreichii

subspecies with adk, rpoB, and fumC genes.

An other type could exist in P. acnes based on gyrB, adk and

rpoB analysis.

Is the discriminatory power of gyrB, rpoB, and adk higher for P.

acnes compared to that of gene recA?

Genetic diversity in P. freudenreichii seems to be more

important than in P. acnes.

15

A good reproducibility was obtained with mass spectrometry:

Identification by mass spectrometry analysis is not dependent

on the equipment and also not dependent on the data analysis

methodology.

Concerning P. acnes, the 3 types previously described were

for a majority also found with mass spectrometry analysis.

Example of CIP 114-09 which has an atypical molecular profile

also has an atypical mass spectrometry profile.

Conclusions (2)

These results underline the strength of the mass

spectrometry method for taxonomy purpose