Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Apr;63(4):1268–1273. doi: 10.1128/aem.63.4.1268-1273.1997

Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

P Kaufmann 1, A Pfefferkorn 1, M Teuber 1, L Meile 1
PMCID: PMC168420  PMID: 9097423

Abstract

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.

Full Text

The Full Text of this article is available as a PDF (487.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bahaka D., Neut C., Khattabi A., Monget D., Gavini F. Phenotypic and genomic analyses of human strains belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. Int J Syst Bacteriol. 1993 Jul;43(3):565–573. doi: 10.1099/00207713-43-3-565. [DOI] [PubMed] [Google Scholar]
  3. Benson D., Lipman D. J., Ostell J. GenBank. Nucleic Acids Res. 1993 Jul 1;21(13):2963–2965. doi: 10.1093/nar/21.13.2963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biavati B., Mattarelli P., Crociani F. Identification of bifidobacteria from fermented milk products. Microbiologica. 1992 Jan;15(1):7–13. [PubMed] [Google Scholar]
  5. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  6. Brunk C. F., Avaniss-Aghajani E., Brunk C. A. A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Appl Environ Microbiol. 1996 Mar;62(3):872–879. doi: 10.1128/aem.62.3.872-879.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crociani F., Biavati B., Alessandrini A., Chiarini C., Scardovi V. Bifidobacterium inopinatum sp. nov. and Bifidobacterium denticolens sp. nov., two new species isolated from human dental caries. Int J Syst Bacteriol. 1996 Apr;46(2):564–571. doi: 10.1099/00207713-46-2-564. [DOI] [PubMed] [Google Scholar]
  8. Embley T. M., Stackebrandt E. The molecular phylogeny and systematics of the actinomycetes. Annu Rev Microbiol. 1994;48:257–289. doi: 10.1146/annurev.mi.48.100194.001353. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Gavini F., Pourcher A. M., Neut C., Monget D., Romond C., Oger C., Izard D. Phenotypic differentiation of bifidobacteria of human and animal origins. Int J Syst Bacteriol. 1991 Oct;41(4):548–557. doi: 10.1099/00207713-41-4-548. [DOI] [PubMed] [Google Scholar]
  11. Gibson G. R., Roberfroid M. B. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995 Jun;125(6):1401–1412. doi: 10.1093/jn/125.6.1401. [DOI] [PubMed] [Google Scholar]
  12. Goldenberger D., Perschil I., Ritzler M., Altwegg M. A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. PCR Methods Appl. 1995 Jun;4(6):368–370. doi: 10.1101/gr.4.6.368. [DOI] [PubMed] [Google Scholar]
  13. Grill J. P., Manginot-Dürr C., Schneider F., Ballongue J. Bifidobacteria and probiotic effects: action of Bifidobacterium species on conjugated bile salts. Curr Microbiol. 1995 Jul;31(1):23–27. doi: 10.1007/BF00294629. [DOI] [PubMed] [Google Scholar]
  14. Langendijk P. S., Schut F., Jansen G. J., Raangs G. C., Kamphuis G. R., Wilkinson M. H., Welling G. W. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl Environ Microbiol. 1995 Aug;61(8):3069–3075. doi: 10.1128/aem.61.8.3069-3075.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lapierre L., Undeland P., Cox L. J. Lithium chloride-sodium propionate agar for the enumeration of bifidobacteria in fermented dairy products. J Dairy Sci. 1992 May;75(5):1192–1196. doi: 10.3168/jds.S0022-0302(92)77866-7. [DOI] [PubMed] [Google Scholar]
  16. Larsen N., Olsen G. J., Maidak B. L., McCaughey M. J., Overbeek R., Macke T. J., Marsh T. L., Woese C. R. The ribosomal database project. Nucleic Acids Res. 1993 Jul 1;21(13):3021–3023. doi: 10.1093/nar/21.13.3021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leblond-Bourget N., Philippe H., Mangin I., Decaris B. 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. Int J Syst Bacteriol. 1996 Jan;46(1):102–111. doi: 10.1099/00207713-46-1-102. [DOI] [PubMed] [Google Scholar]
  18. Ludwig W., Schleifer K. H. Bacterial phylogeny based on 16S and 23S rRNA sequence analysis. FEMS Microbiol Rev. 1994 Oct;15(2-3):155–173. doi: 10.1111/j.1574-6976.1994.tb00132.x. [DOI] [PubMed] [Google Scholar]
  19. Mangin I., Bourget N., Decaris B. Ribosomal DNA polymorphism in the genus Bifidobacterium. Res Microbiol. 1996 Mar-Apr;147(3):183–192. doi: 10.1016/0923-2508(96)80218-x. [DOI] [PubMed] [Google Scholar]
  20. Mangin I., Bourget N., Simonet J. M., Decaris B. Selection of species-specific DNA probes which detect strain restriction polymorphism in four Bifidobacterium species. Res Microbiol. 1995 Jan;146(1):59–71. doi: 10.1016/0923-2508(96)80271-3. [DOI] [PubMed] [Google Scholar]
  21. Neefs J. M., Van de Peer Y., De Rijk P., Chapelle S., De Wachter R. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res. 1993 Jul 1;21(13):3025–3049. doi: 10.1093/nar/21.13.3025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Olsen G. J., Woese C. R., Overbeek R. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol. 1994 Jan;176(1):1–6. doi: 10.1128/jb.176.1.1-6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rice C. M., Fuchs R., Higgins D. G., Stoehr P. J., Cameron G. N. The EMBL data library. Nucleic Acids Res. 1993 Jul 1;21(13):2967–2971. doi: 10.1093/nar/21.13.2967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rijpens N. P., Jannes G., Van Asbroeck M., Rossau R., Herman L. M. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl Environ Microbiol. 1996 May;62(5):1683–1688. doi: 10.1128/aem.62.5.1683-1688.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wang R. F., Cao W. W., Cerniglia C. E. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl Environ Microbiol. 1996 Apr;62(4):1242–1247. doi: 10.1128/aem.62.4.1242-1247.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yamamoto T., Morotomi M., Tanaka R. Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora. Appl Environ Microbiol. 1992 Dec;58(12):4076–4079. doi: 10.1128/aem.58.12.4076-4079.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES