Evaluation of the diversity of two species of the genus Propionibacterium : Mass Spectrometry versus Triple-Locus Sequence Analysis F. Valence 1, P. Bouvet 2, J. Jardin 3, M. Dalmasso 3, L. Motreff 4, C. Pissis 4, S. Lortal 3, C. Bizet 4 and D. Clermont 4 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
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 2
Propionibacterium freudenreichii and Propionibacterium acnes: Habitat and Functions 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 ) 3
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) 4
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) Origin P. freudenreichii subsp. freudenreichii P. freudenreichii subsp. shermanii raw milk / 6 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 tly genes: Types I, II, III Origin P. acnes blood 7 cheese 1 8 Various (acne, abscess..) 9 "vegetal" (hay, straw, wheat) / 4 unknown / 1 unknown 5 5
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 (Wahl et al. 1999) Bruker Daltonics autoflex (IP): Comparison with high quality reference spectra, generated by culture collections Applied Biosystems equipment (INRA/CIRM): Statistical analysis of the data: 6 Principal component analysis
Results: Phylogenetic tree based on 16S rrna gene sequences for P. acnes 0.01 P.Acnes type I 108-02 228-08 391-00 139-09 Type I 53-117T P.acnes type I 219-08 A179 54.158 60.42 217-02 53.119 266-02 483-03 P.Acnes type I P.Acnes type III P.Acnes type III P.Acnes type III 62 P.Acnes type III P.Acnes type III 81-02 P.Acnes type II 27-08 99 335-02 110-06 Type II 60 P.Acnes type II P.Acnes type II 55 329-00 511-00 71-01 114-09 103261T Propionibacterium avidum 105357T Actinomyces turicensis Type III - A. turencis was used as outgroup. Neighbourg-joining method. - Numbers represent bootstrap values of replicates - 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 7
Phylogenetic tree based on combinated adk, rpob and gyrb gene sequences for P. acnes Type I Type III Type II 0.002 88 99 CIP 60-42 391-00 266-02 CIP 53-119 217-02 CIP 53-117T 483-03 CIP 54-158 81-02 15 52 35 71-01 108-02 139-09 228-08 CIP A179 - The 3 types are well defined - Type I contains 2 groups not seen with 16S rrna and not already described. - No relation between strain origins and clusters. Colour Code : blood, various, unknown 27-08 63 110-06 335-02 219-08 329-00 - All the old strains belong to Type I 8 - No Type specificity for the strains recently isolated.
67 Phylogenetic trees based on combinated adk, rpob and fumc gene sequences for P. freudenreichii CIRM-BIA683 CIRM-BIA456 CIRM-BIA514 TL600 CIRM-BIA141 CIRM-BIA516 CIRM-BIA515 CIRM-BIA686 CIRM-BIA527 63 77 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-BIA121 P. freudenreichii subsp. freudenreichii CIRM-BIA693 CIRM-BIA7 CIRM-BIA513 CIRM-BIA508 CIRM-BIA1T P. freudenreichii subsp. shermanii CIRM-BIA688 CIRM-BIA689 99 CIRM-BIA692 CIRM-BIA8 CIRM-BIA512 - No relation between strain origin and clusters. Colour Code : Cheese, raw milk, wheat Results confirmed by MLST study of 7 genes on strains. See Area 2 - Poster Number: 17 "Lineages with broad dairy biotope ranges and phenotypic variability in Propionibacterium freudenreichii revealed by multilocus sequence typing" 9
P. freudenreichii +TOF MS: 60 MCA scans from BIA121_25.wiff a=3.57326353521455530e-004, t0=-3.37680250247140070e+001 138 135 130 125 120 115 110 105 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 2600.5916 4421.3684 3866.2730 5183.0350 5650.2760 6063.4253 6532.6797 7042.8393 6888.0561 8418.6259 8988.0516 9545.0821 10211.8612 10878.3449 6953.8237 88.8548 6502.4825 7816.2918 10439.4645 5139.1079 6700.7972 8829.1876 5686.1622 6808.9025 7658.4276 8046.4845 10406.2777 13534.2838 Max. 138.0 counts. 0 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 1.6e4 m/z, amu +TOF MS: 60 MCA scans from BIA141_25.wiff Max. 114.0 counts. a=3.57326353521455530e-004, t0=-3.37680250247140070e+001 114 110 105 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 2600.6004 2537.4684 2697.5995 3867.3105 3296.8610 5651.0369 6041.5463 4423.4146 6021.4075 5185.1722 4057.2767 5139.9518 6417.9264 5038.0748 6576.7373 4683.7171 7239.8068 7012.8622 6812.3445 6533.5592 6888.0482 7356.3042 7245.5587 8222.7988 7860.4448 8419.6725 8422.5274 9545.5076 10150.7743 8988.0449 7842.5275 6709.8038 9504.3040 7095.6173 8968.1840 7416.7621 7552.0599 9598.5488 10877.3349 8032.2679 9336.6367 73.4388 13808.7165 14914.4271 0 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 1.6e4 +TOF MS: 60 MCA scans from BIA514_50.wiff m/z, amu Max. 131.0 counts. a=3.57326353521455530e-004, t0=-3.37680250247140070e+001 130 125 120 115 110 105 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 3867.4143 4420.5742 5183.0641 5648.0778 6532.6883 7012.9668 7241.1422 6888.2029 7861.4903 6812.7703 6514.3623 2600.5970 7279.7986 2402.6626 5086.8907 6101.5899 5051.7851 4458.3251 6185.7424 7746.8215 8419.7723 8989.0085 8204.0703 9546.3601 7526.9201 9180.0097 10212.6103 10133.7225 9339.2071 9565.0476 10425.2204 10878.6293 14932.7149 0 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 1.6e4 m/z, amu P. acnes +TOF MS: 60 MCA scans from P228-08_50.wiff a=3.57327121350785870e-004, t0=-3.36406827176178920e+001 376 360 340 320 300 280 260 240 220 200 180 160 140 120 80 60 40 20 2601.8348 4371.3740 5168.8165 7180.0331 7001.4589 9589.5122 Max. 376.0 counts. 0 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 1.6e4 +TOF MS: 60 MCA scans from P219-08_25.wiff m/z, amu Max. 358.0 counts. a=3.57327121350785870e-004, t0=-3.36406827176178920e+001 358 340 320 300 280 260 240 220 200 180 160 140 120 80 60 40 20 0 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 1.6e4 +TOF MS: 60 MCA scans from P27-08_75.wiff m/z, amu Max. 226.0 counts. a=3.57327121350785870e-004, t0=-3.36406827176178920e+001 226 220 210 200 190 180 170 160 150 140 130 120 110 90 80 70 60 50 40 30 20 10 2600.7383 4372.8862 4373.4747 5170.7827 7179.5818 6986.4498 7251.2177 6985.0787 6785.6615 6576.3208 3942.0568 8667.1882 9589.4848 9589.2980 9547.2606 0 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 1.6e4 m/z, amu MALDI-TOF results: examples of profiles BIA_121 P228-08 In te n s ity, c o u n ts 7859.6347 8222.8489 In te n s ity, c o u n ts BIA_141 In te n s ity, c o u n ts P219-08 In te n s ity, c o u n ts 2601.5938 5168.9323 6806.8667 7256.5195 In te n s ity, c o u n ts 6064.5849 7043.9160 8224.5297 BIA_514 In te n s ity, c o u n ts P27-08 2 types of profiles in relation with the species of Propionibacterium One species = one specific profile 10
MALDI-TOF results: Principal Component Analysis 12 spectra obtained for each strain 2 groups corresponding to the 2 species P. freudenreichii and P. acnes 11
MALDI-TOF results: Principal Component Analysis: strain 114-09 12 spectra obtained for each strain Strain 114-09 : away from other strains according to phylogenetic trees based on 16S 12 rrna and genes rpob, gyrb and adk, it also shows an atypical mass profil
MALDI-TOF results: Comparison between the two equipments used MSP Dendrogram P. acnes P. freudenreichii 0900 800 700 600 500 400 300 200 Distance Level 114.09 P. acnes 511.00 P. acnes 335.02 P. acnes 81.02 P. acnes 27.08 P. acnes 110.06 P. acnes CIP 53.119 P. acnes CIP 53.117 T P. acnes CIP 60.42 P. acnes CIP 54.158 P. acnes 266.02 P. acnes 483.03 P. acnes 228.08 P. acnes 219.08 P. acnes CIP A179 P. acnes 139.09 P. acnes 229.08 P. acnes 71.01_P. acnes 108.02 P.acnes 1 P. freudenreichii 516 P. freudenreichii 513 P._freudenreichii 508 P. freudenreichii 688 P. freudenreichii 686 P. freudenreichii 683 P. freudenreichii 8 P. freudenreichii 515 P. freudenreichii 512 P. freudenreichii 7 P. freudenreichii 527 P. freudenreichii 514 P. freudenreichii 693 P. freudenreichii TL600 P.freudenreichii 689 P. freudenreichii 0 Type II and III Type I P. acnes P. freudenreichii Applied Biosystems equipment (INRA/CIRM) Bruker Daltonics autoflex (IP) - Similar results obtained whathever the equipment and analyse type performed 13 - Regarding P. acnes, the groups obtained obviously reflect the molecular types
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. 14
Conclusions (2) 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. These results underline the strength of the mass spectrometry method for taxonomy purpose 15