Antonie van Leeuwenhoek

, Volume 112, Issue 12, pp 1785–1800 | Cite as

Genetic marker-based multi-locus sequence analysis for classification, genotyping, and phylogenetics of the family Bifidobacteriaceae as an alternative approach to phylogenomics

  • Chahrazed Mekadim
  • Věra Bunešová
  • Eva Vlková
  • Zuzana Hroncová
  • Jiří KillerEmail author
Original Paper


Bifidobacteria are widely known for their probiotic potential; however, little is known regarding the ecological significance and potential probiotic effects of the phylogenetically related ‘scardovial’ genera (Aeriscardovia, Alloscardovia, Bombiscardovia, Galliscardovia, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia) and Gardnerella classified with bifidobacteria within the Bifidobacteriaceae family. Accurate classification and genotyping of bacteria using certain housekeeping genes is possible, whilst current phylogenomic analyses allow for extremely precise classification. Studies of applicable genetic markers may provide results comparable to those obtained from phylogenomic analyses of the family Bifidobacteriaceae. Segments of the glyS (624 nucleotides), pheS (555 nucleotides), rpsA (630 nucleotides), and rpsB (432 nucleotides) genes and their concatenated sequence were explored. The mean glyS, pheS, rpsB and rpsA gene sequence similarities calculated for Bifidobacterium taxa were 84.8, 85.2, 90.2 and 86.8%, respectively. Interestingly, the average value of the Average Nucleotide Identity among 67 type strains of the family Bifidobacteriaceae (84.70%) calculated based on values published recently was in agreement with the average pairwise similarity (84.6%) among 75 type strains of Bifidobacteriaceae family computed in this study using the concatenated sequences of four gene fragments. Similar to phylogenomic analyses, several gene sequence and phylogenetic analyses revealed that concatenated gene regions allow for classification of Bifidobacteriaceae strains into particular phylogenetic clusters and groups. Phylogeny reconstructed from the concatenated sequences assisted in defining two novel phylogenetic groups, the Bifidobacterium psychraerophilum group consisting of B. psychraerophilum, Bifidobacterium crudilactis and Bifidobacterium aquikefiri species and the Bifidobacterium bombi group consisting of B. bombi, Bifidobacterium bohemicum and Bifidobacterium commune.


Bifidobacteria Scardovia Identification Housekeeping genes 



This study was supported by Project Excellence [Grant No. CZ.02.1.01/0.0/0.0/15_003/0000460], the Czech National Agency for Agricultural Research [Projects Nos. QJ1610248 and QJ1510338], and an internal university grant application [Grant No. CIGA 20182018].

Author’s contribution

JK, CM are conceived and designed experiments. CM, VB, EV, ZH, JK are performed the experiments. JK, VB, EV, CM are analysed the data. VB, EV, ZH are contributed reagents/materials/analysis tools. JK, CM are wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they no conflict of interest.

Supplementary material

10482_2019_1307_MOESM1_ESM.pptx (1.8 mb)
Supplementary material 1 (PPTX 1847 kb)
10482_2019_1307_MOESM2_ESM.xlsx (32 kb)
Supplementary material 2 (XLSX 31 kb)
10482_2019_1307_MOESM3_ESM.xls (116 kb)
Supplementary material 3 (XLS 115 kb)


  1. Biavati B, Mattarelli P (2018) Related genera within the family Bifidobacteriaceae. In: Biavati B, Mattarelli P, Holzapfel WH, Wood BJB (eds) The bifidobacteria and related organisms: biology, taxonomy, applications. Academic Press, London, p 49Google Scholar
  2. Bottacini F, Morrissey R, Esteban-Torres M, James K, van Breen J, Dikareva E, Egan M, Lambert J, van Limpt K, Knol J, O’Connell Motherway M, van Sinderen D (2018) Comparative genomics and genotype-phenotype associations in Bifidobacterium breve. Sci Rep 8:10633PubMedPubMedCentralGoogle Scholar
  3. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S, Trujillo ME (2018) Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68:461–466Google Scholar
  4. Delétoile A, Passet V, Aires J, Chambaud I, Butel MJ, Smokvina T, Brisse S (2010) Species delineation and clonal diversity in four Bifidobacterium species as revealed by multilocus sequencing. Res Microbiol 161:82–90PubMedGoogle Scholar
  5. Duan H, Liu G, Wang X, Fu Y, Liang Q, Shang Y, Chu N, Huang H (2015) Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification. Biomed Res Int 2015:271728PubMedPubMedCentralGoogle Scholar
  6. Duranti S, Milani C, Lugli GA, Turroni F, Mancabelli L, Sanchez B, Ferrario C, Viappiani A, Mangifesta M, Mancino W, Gueimonde M, Margolles A, van Sinderen D, Ventura M (2015) Insights from genomes of representatives of the human gut commensal Bifidobacterium bifidum. Environ Microbiol 17:2515–2531PubMedGoogle Scholar
  7. Glaeser SP, Kämpfer P (2015) Multilocus sequence analysis (MLSA) in prokaryotic taxonomy. Syst Appl Microbiol 38:237–245PubMedGoogle Scholar
  8. Killer J, Kopečný J, Mrázek J, Havlík J, Koppová I, Benada O, Rada V, Kofroňová O (2010) Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees. Syst Appl Microbiol 33:359–366PubMedGoogle Scholar
  9. Killer J, Mrázek J, Bunešová V, Havlík J, Koppová I, Benada O, Rada V, Kopečný J, Vlková E (2013a) Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa). Syst Appl Microbiol 36:11–16PubMedGoogle Scholar
  10. Killer J, Ročková Š, Vlková E, Rada V, Havlík J, Kopecny J, Bunesová V, Benada O, Kofronová O, Pechar R, Profousová I (2013b) Alloscardovia macacae sp. nov., isolated from the milk of a macaque (Macaca mulatta), emended description of the genus Alloscardovia and proposal of Alloscardovia criceti comb. nov. Int J Syst Evol Microbiol 63:4439–4446PubMedGoogle Scholar
  11. Killer J, Sedláček I, Rada V, Havlík J et al (2013c) Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis. Int J Syst Evol Microbiol 63:4350–4353PubMedGoogle Scholar
  12. Killer J, Mekadim C, Pechar R, Bunešová V, Mrázek J, Vlková E (2018a) Gene encoding the CTP synthetase as an appropriate molecular tool for identification and phylogenetic study of the family Bifidobacteriaceae. MicrobiologyOpen 7:e00579PubMedPubMedCentralGoogle Scholar
  13. Killer J, Mekadim C, Pechar R, Bunešová V, Vlková E (2018b) The threonine-tRNA ligase gene region is applicable in classification, typing, and phylogenetic analysis of bifidobacteria. J Microbiol 56:713–721PubMedGoogle Scholar
  14. Leclerque A, Hartelt K, Schuster C, Jung K, Kleespies RG (2011) Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria. FEMS Microbiol Lett 324:125–134PubMedGoogle Scholar
  15. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452Google Scholar
  16. Lugli GA, Milani C, Turroni F, Duranti S, Ferrario C, Viappiani A, Mancabelli L, Mangifesta M, Taminiau B, Delcenserie V, van Sinderen D, Ventura M (2014) Investigation of the evolutionary development of the genus Bifidobacterium by comparative genomics. Appl Environ Microbiol 80:6383–6394PubMedPubMedCentralGoogle Scholar
  17. Lugli GA, Milani C, Turroni F, Duranti S, Mancabelli L, Mangifesta M, Ferrario C, Modesto M, Mattarelli P, Killer J, van Sinderen D, Ventura M (2017) Comparative genomic and phylogenomic analyses of the Bifidobacteriaceae family. BMC Genom 18:568Google Scholar
  18. Lugli GA, Mancino W, Milani C, Duranti S, Turroni F, van Sinderen D, Ventura M (2018a) Reconstruction of the bifidobacterial pan-secretome reveals the network of extracellular interactions between bifidobacteria and the infant gut. Appl Environ Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lugli GA, Mangifesta M, Duranti S, Anzalone R, Milani C, Mancabelli L, Alessandri G, Turroni F, Ossiprandi MC, van Sinderen D, Ventura M (2018b) Phylogenetic classification of six novel species belonging to the genus Bifidobacterium comprising Bifidobacterium anseris sp. nov., Bifidobacterium criceti sp. nov., Bifidobacterium imperatoris sp. nov., Bifidobacterium italicum sp. nov., Bifidobacterium margollesii sp. nov. and Bifidobacterium parmae sp. nov. Syst Appl Microbiol 41:173–183PubMedGoogle Scholar
  20. Lugli GA, Milani C, Duranti S, Mancabelli L, Mangifesta M, Turroni F, Viappiani A, van Sinderen D, Ventura M (2018c) Tracking the taxonomy of the genus Bifidobacterium based on a phylogenomic approach. Appl Environ Microbiol 84(4):pii: e02249-17. CrossRefGoogle Scholar
  21. Martens M, Weidner S, Linke B, de Vos P, Gillis M, Willems A (2007) A prototype taxonomic microarray targeting the rpsA housekeeping gene permits species identification within the rhizobial genus Ensifer. Syst Appl Microbiol 30:390–400PubMedGoogle Scholar
  22. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P (2010) RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26:2462–2463PubMedPubMedCentralGoogle Scholar
  23. Mekadim C, Killer J, Mrázek J, Bunešová V, Pechar R, Hroncová Z, Vlková E (2018a) Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales. Arch Microbiol 200:1427–1437PubMedGoogle Scholar
  24. Mekadim C, Killer J, Pechar R, Mrázek J (2018b) Variable regions of the glyS, infB and rplB genes usable as novel genetic markers for identification and phylogenetic purposes of genera belonging to the family Propionibacteriaceae. Int J Syst Evol Microbiol 68:2697–2705PubMedGoogle Scholar
  25. Naser SM, Thompson FL, Hoste B, Gevers D, Dawyndt P, Vancanneyt M, Swings J (2005) Application of multilocus sequence analysis for rapid identification of Enterococcus species on rpoA and pheS genes. Microbiology 151:2141–2150PubMedGoogle Scholar
  26. Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Vancanneyt M, Swings J (2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 57:2777–2789PubMedGoogle Scholar
  27. Naushad HS, Gupta RS (2013) Phylogenomics and molecular signatures for species from the plant pathogen-containing order xanthomonadales. PLoS ONE 8:e55216PubMedPubMedCentralGoogle Scholar
  28. Nouioui I, Carro L, García-López M, Meier-Kolthoff JP, Woyke T, Kyrpides NC, Pukall R, Klenk HP, Goodfellow M, Göker M (2018) Genome-Based Taxonomic Classification of the Phylum Actinobacteria. Front Microbiol 9:2007PubMedPubMedCentralGoogle Scholar
  29. Novichkov PS, Wolf YI, Dubchak I, Koonin EV (2009) Trends in prokaryotic evolution revealed by comparison of closely related bacterial and archaeal genomes. J Bacteriol 191:65–73PubMedGoogle Scholar
  30. Pechar R, Killer J, Salmonová H, Geigerová M, Švejstil R, Švec P, Sedláček I, Rada V, Benada O (2017a) Bifidobacterium apri sp. nov., a thermophilic actinobacterium isolated from the digestive tract of wild pigs (Sus scrofa). Int J Syst Evol Microbiol 67:2349–2356PubMedGoogle Scholar
  31. Pechar R, Killer J, Švejstil R, Salmonová H, Geigerová M, Bunešová V, Rada V, Benada O (2017b) Galliscardovia ingluviei gen. nov., sp. nov., a thermophilic bacterium of the family Bifidobacteriaceae isolated from the crop of a laying hen (Gallus gallus f. domestica). Int J Syst Evol Microbiol 67:2403–2411PubMedGoogle Scholar
  32. Rada V, Petr J (2000) A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca. J Microbiol Methods 43:127–132PubMedGoogle Scholar
  33. Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131PubMedGoogle Scholar
  34. Russell DA, Ross RP, Fitzgerald GF, Stanton C (2011) Metabolic activities and probiotic potential of bifidobacteria. Int J Food Microbiol 149:88–105PubMedGoogle Scholar
  35. Sanders ME, Benson A, Lebeer S, Merenstein DJ, Klaenhammer TR (2018) Shared mechanisms among probiotic taxa: implications for general probiotic claims. Curr Opin Biotechnol 49:207–216PubMedGoogle Scholar
  36. Simůnek J Jr, Killer J, Sechovcová H, Šimůnek J, Pechar R, Rada V, Švec P, Sedláček I (2018) Characterization of a xylanolytic bacterial strain C10 isolated from the rumen of a red deer (Cervus elaphus) closely related of the recently described species Actinomyces succiniciruminis, A. glycerinitolerans, and A. ruminicola. Folia Microbiol (Praha) 63:391–399Google Scholar
  37. Sun Z, Zhang W, Guo C, Yang X, Liu W, Wu Y, Song Y, Kwok LY, Cui Y, Menghe B, Yang R, Hu L, Zhang H (2015) Comparative genomic analysis of 45 type strains of the genus Bifidobacterium: a snapshot of its genetic diversity and evolution. PLoS ONE 10:e0117912PubMedPubMedCentralGoogle Scholar
  38. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedPubMedCentralGoogle Scholar
  39. Tojo R, Suárez A, Clemente MG, de los Reyes-Gavilán CG, Margolles A, Gueimonde M, Ruas-Madiedo P (2014) Intestinal microbiota in health and disease: role of bifidobacteria in gut homeostasis. World J Gastroenterol 20:15163–15176PubMedPubMedCentralGoogle Scholar
  40. Tomasini N, Lauthier JJ, Llewellyn MS, Diosque P (2013) MLSTest: novel software for multi-locus sequence data analysis in eukaryotic organisms. Inf Gen Evol 20:188–196Google Scholar
  41. Vaneechoutte M, Guschin A, Van Simaey L, Gansemans Y, Van Nieuwerburgh F, Cools P (2018) Emended description of Gardnerella vaginalis and description of Gardnerella leopoldii sp. nov., Gardnerella piotii sp. nov. and Gardnerella swidsinskii sp. nov., with delineation of 13 genomic species within the genus Gardnerella. Int J Syst Evol Microbiol 69:679–687Google Scholar
  42. Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R (2003) Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69:6908–6922PubMedPubMedCentralGoogle Scholar
  43. Ventura M, Canchaya C, Del Casale A, Dellaglio F, Neviani E, Fitzgerald GF, van Sinderen D (2006) Analysis of bifidobacterial evolution using a multilocus approach. Int J Syst Evol Microbiol 56:2783–2792PubMedGoogle Scholar
  44. Ventura M, Turroni F, Zomer A, Foroni E, Giubellini V, Bottacini F, Canchaya C, Claesson MJ, He F, Mantzourani M, Mulas L, Ferrarini A, Gao B, Delledonne M, Henrissat B, Coutinho P, Oggioni M, Gupta RS, Zhang Z, Beighton D, Fitzgerald GF, O’Toole PW, van Sinderen D (2009) The Bifidobacterium dentium Bd1 genome sequence reflects its genetic adaptation to the human oral cavity. PLoS Genet 5:e1000785PubMedPubMedCentralGoogle Scholar
  45. Verma M, Lal D, Kaur J, Saxena A, Kaur J, Anand S, Lal R (2013) Phylogenetic analyses of phylum Actinobacteria based on whole genome sequences. Res Microbiol 164:718–728PubMedGoogle Scholar
  46. von Ah U, Mozzetti V, Lacroix C, Kheadr EE, Fliss I, Meile L (2007) Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum. BMC Microbiol 7:79Google Scholar
  47. Wang Z, Wu M (2013) A phylum-level bacterial phylogenetic marker database. Mol Biol Evol 30:1258–1262PubMedGoogle Scholar
  48. Wu D, Jospin G, Eisen JA (2013) Systematic identification of gene families for use as “markers” for phylogenetic and phylogeny-driven ecological studies of bacteria and archaea and their major subgroups. PLoS ONE 8:e77033PubMedPubMedCentralGoogle Scholar
  49. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613–1617PubMedPubMedCentralGoogle Scholar
  50. Zhang G, Gao B, Adeolu M, Khadka B, Gupta RS (2016) Phylogenomic analyses and comparative studies on genomes of the Bifidobacteriales: identification of molecular signatures specific for the order Bifidobacteriales and its different subclades. Front Microbiol 7:978PubMedPubMedCentralGoogle Scholar
  51. Zhi XY, Li WJ, Stackebrandt E (2009) An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol 59:589–608PubMedGoogle Scholar
  52. Zwick A, Regier JC, Zwickl DJ (2012) Resolving discrepancy between nucleotides and amino acids in deep-level arthropod phylogenomics: differentiating serine codons in 21-amino-acid models. PLoS ONE 7:e47450PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Institute of Animal Physiology and Genetics of the Czech Academy of SciencesKrčCzech Republic
  2. 2.Department of Microbiology, Nutrition and Dietetics, Food and Natural Resources, Faculty of AgrobiologyCzech University of Life SciencesSuchdolCzech Republic

Personalised recommendations