Skip to main content
SpringerLink
Log in

Sequence analysis of 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region for differentiation of probioticsLactobacillus strains isolated from the gastrointestinal tract of chicken

  • Systematics/Taxonomy
  • Original Articles
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

Twelve probioticLactobacillus strains which were previously identified with classical biochemical tests were re-identified using molecular methods. Comparative sequence analyses of the 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region (ISR) were applied. Results of the study showed that mis-identification at species level occurred at high rate when classical biochemical tests were used. Nine of the strains showed discrepancy in their identity. These nine strains which were previously identified through biochemical tests asL. brevis C1,L. brevis C10,L. fermentum C16,L. brevis C17,L. crispatus I12,L. acidophilus I16,L. fermentum I24,L. fermentum I25 andL. acidophilus I26 were re-identified asL. reuteri C1,L. reuteri C10,L. reuteri C16,L. panis C17,L. brevis I12,L. gallinarum I16,L. salivarius I24,L. brevis I25 andL. gallinarum I26, respectively, using 16S rRNA gene and 16S–23S rRNA gene ISR analysis.Lactobacillus strains I16 and I26 initially could not be classified into a single taxon by 16S rRNA gene sequencing but the identities of these two strains were eventually resolved by 16S–23S rRNA gene ISR sequence analysis asL. gallinarum. Sequence analysis of 16S rRNA gene in complementary with 16S–23S rRNA gene ISR could be potentially useful for rapid and reliable identification of bacteria.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altschul F.S., Gish W., Miller W., Myers E.W., Lipman D.J. (1990). Basic Local Alignment Search Tool. J. Mol. Biol., 215: 403–410.

    CAS  PubMed  Google Scholar 

  • Amann R.I., Ludwig W., Schleifer K.H. (1995). Phylogenetic identification andin situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59: 143–169.

    CAS  PubMed  Google Scholar 

  • Ashelford K.E., Chuzhanova N., Fry J.C., Jones A.J., Weightman A.J. (2005). At least one in twenty 16S rRNA sequence records currently held in public repositories estimated to contain substantial anomalies. Appl. Environ. Microbiol., 71: 7724–7736.

    Article  CAS  PubMed  Google Scholar 

  • Benson D.A., Karsch-Mizrachi I., Lipman D.J., Ostell J., Wheeler D.L. (2005). GenBank. Nucl. Acid. Res., 33: D34-D38.

    Article  CAS  Google Scholar 

  • Berthier F., Ehrlich S.D. (1998). Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Micrbiol. Lett., 161: 97–106.

    Article  CAS  Google Scholar 

  • Bosshard P.P., Abels S., Altwegg M., Böttger E.C., Zbinden R. (2004). Comparison of conventional and molecular methods for identification of aerobic catalase-negative Gram-positive cocci in the clinical laboratory. J. Clin. Microbiol., 42: 2065–2073.

    Article  CAS  PubMed  Google Scholar 

  • Bosshard P.P., Zbinden R., Abels S., Böddinghaus B., Altwegg M., Böttger E.C. (2006). 16S rRNA Gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory. J. Clin. Microbiol., 44: 1359–1366.

    Article  CAS  PubMed  Google Scholar 

  • Boyd M.A., Antonio M.A.D., Hiller S.L. (2005). Comparison of API 50 CH strips to whole-chromosomal DNA probes for identification ofLactobacillus species. J. Clin. Microbiol., 43: 5309–5311.

    Article  CAS  PubMed  Google Scholar 

  • Chavagnat F., Hauter M., Jimeno J., Casey M.G. (2002). Comparison of partial tuf gene sequences for the identification of lactobacilli. FEMS Microbiol. Lett., 217: 177–183.

    Article  CAS  PubMed  Google Scholar 

  • Chevallier B., Hubert J.-C., Kammerer B. (1994). Determination of chromosome size and number of rrn loci inLactobacillus plantarum by pulse-field gel electrophoresis. FEMS Micrbiol. Lett., 120: 51–56.

    Article  CAS  Google Scholar 

  • Clayton R.A., Sutton G., Hinkle J.P.S., Bult C., Fields C. (1995). Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa. Int. J. Syst. Bacteriol., 45: 595–599.

    CAS  PubMed  Google Scholar 

  • Curtis T.P., Sloan W.T., Scannell J.W. (2002). Estimating prokaryotic diversity and its limits. Proc. Natl. Acad. Sci. USA, 99: 10494–10499.

    Article  CAS  PubMed  Google Scholar 

  • Felis G.E., Dellaglio F. (2007). Taxonomy of Lactobacilli and Bifidobacteria. Curr. Issues Intest. Microbiol., 8: 44–61.

    CAS  PubMed  Google Scholar 

  • Felsenstein J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39: 783–791.

    Article  Google Scholar 

  • Flint J.F., Angert E.R. (2005). Development of a strain-specific assay for detection of viableLactobacillus sp. HOFG1 after application to cattle feed. J. Microbiol. Methods, 61: 235–243.

    Article  CAS  PubMed  Google Scholar 

  • Fuller R. (1995). Probiotics: Prospects of use in opportunistic infections. In: Fuller R., Heidt P.J., Rusch V., van deer Waaij D., Eds, Probiotics: Their Development and Use, Herborn-Dill, Germany, pp. 1–7.

  • Gürtler V., Stanisich V.A. (1996). New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology, 142: 3–16.

    Article  PubMed  Google Scholar 

  • Hall T.A. (1999). Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp., 41: 95–98.

    CAS  Google Scholar 

  • Hensiek R., Krupp G., Stackebrandt E. (1992). Development of diagnostic oligonucleotide probes for fourLactobacillus species occurring in the intestinal tract. Syst. Appl. Bacteriol., 15: 123–128.

    CAS  Google Scholar 

  • Huber T., Faulker G., Hugenholtz P. (2004). Ballerophon; a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics, 20: 2317–2319.

    Article  CAS  PubMed  Google Scholar 

  • Jensen M.A., Webster J.A., Straus N. (1993). Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol., 59: 945–952.

    CAS  PubMed  Google Scholar 

  • Jin L.Z., Ho Y.W., Ali M.A., Abdullah N., Ong K.B., Jalaludin S. (1996). Adhesion ofLactobacillus isolates to intestinal epithelial cells of chicken. Lett. Appl. Microbiol., 22: 229–232.

    Article  CAS  PubMed  Google Scholar 

  • Jin L.Z., Ho Y.W., Abdullah N., Jalaludin S. (1998). Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets containingLactobacillus cultures. Poultry Sci., 77: 1259–1265.

    CAS  Google Scholar 

  • Jin L.Z., Ho Y.W., Abdullah N., Jalaludin S. (2000). Digestive and bacterial enzyme activities in broilers fed diets supplemented withLactobacillus culture. Poultry Sci., 77: 1259–1265.

    Google Scholar 

  • Jukes T.H., Cantor C.R. (1969). Mammalian protein metabolism. In: Munro H.N., Ed., Evolution of Protein Molecules. Academic Press, London, pp. 21–132.

    Google Scholar 

  • Kalavathy R., Abdullah N., Jalaludin S., Ho Y.W. (2003). Effects ofLactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. Brit. Poultry Sci., 79: 886–891.

    Google Scholar 

  • Kumar S., Tamura K., Nei M. (2004). MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinformatics, 5: 150–163.

    Article  CAS  PubMed  Google Scholar 

  • Leblond-Bourget N., Philippe H., Mangin I., Decaris B. (1996). 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecificBifidobacterium phylogeny. Int. Syst. Bacteriol., 46: 102–111.

    Article  CAS  Google Scholar 

  • Li Y., Raftis E., Canchaya C., Fitzgerald G.F., van Sinderen D., O’Toole P.W. (2006). Polyphasic analysis indicates thatLactobacillus salivarius subsp.salivarius andLactobacillus salivarius subsp.salicinius do not merit separate subspecies status. Int. J. Syst. Evol. Microbiol., 56: 2397–2403.

    Article  CAS  PubMed  Google Scholar 

  • Ludwig W., Schleifer K. (1999). Phylogeny of bacteria beyond the 16S rRNA standard. ASM News, 65: 752–757.

    Google Scholar 

  • Mättö J., Fondén R., Tolvanen T., von Wright A., Vilpponen-Salmela T., Satokari R., Saarela M. (2006). Intestinal survival and persistence of probioticLactobacillus andBifidobacterium strains administered in triple-strain yoghurt. Int. Dairy J., 16: 1174–1180.

    Article  CAS  Google Scholar 

  • Montesi A., García-Albiach R., Pozuelo M.J., Pintado C., Goñi I., Rotger R. (2005). Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics. Int. J. Food Microbiol., 98: 281–289.

    Article  CAS  PubMed  Google Scholar 

  • Nour M. (1998). 16S–23S and 23S–5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis. Res. Microbiol., 149: 433–448.

    Article  CAS  PubMed  Google Scholar 

  • Quadghiri M., Amar M., Vancanneyt M., Swings J. (2005). Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben). FEMS Microbiol. Lett., 251: 267–271.

    Article  CAS  Google Scholar 

  • Rosselló-Mora R., Amann R. (2001). The species concept for prokaryotes. FEMS Microbiol. Lett., 25: 39–67.

    Google Scholar 

  • Roussel Y., Colmin C., Simonet J.M., Decaris B. (1993). Strain characterization genome size and plasmid content in theLactobacillus acidophilus group (Hansen and Mocquot). J Appl. Bacteriol., 74: 549–556.

    CAS  PubMed  Google Scholar 

  • Saitou N., Nei M. (1987). The Neighbour-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4: 406–425.

    CAS  PubMed  Google Scholar 

  • Sánchez I., Seseña S., Palop L. (2003). Identification of lactic acid bacteria from spontaneous fermentation of ‘Almagro’ eggplants by SDS-PAGE whole cell protein fingerprinting. Int. J. Food Microbiol., 82: 181–189.

    Article  PubMed  Google Scholar 

  • Simmering R., Blaut M. (2001). Pro- and pre-biotics — the tasty guardian angels? Appl. Microbiol. Biotechnol., 55: 19–28.

    Article  CAS  PubMed  Google Scholar 

  • Snell-Castro R., Godon J.-J., Delgenès J.-P., Dabert P. (2005). Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis. FEMS Microbiol. Ecol., 52: 229–242.

    Article  CAS  PubMed  Google Scholar 

  • Song Y.-L., Kato N., Matsumiya Y., Liu C.-X., Kato H., Watanabe K. (1999). Identification ofLactobacillus species of human origin by a commercial kit API 50 CHL. J. Rapid Methods Autom. Microbiol., 10: 77–82.

    CAS  Google Scholar 

  • Stackebrandt E., Goebel B.M. (1994). Taxonomy note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol., 28.

  • Stoyancheva G.D., Danova T.S., Boudakov I.Y. (2006). Molecular identification of vaginal lactobacilli isolated from Bulgarian women. Antonie van Leeuwenhoek, 90: 201–210.

    Article  CAS  PubMed  Google Scholar 

  • Tamang P.J., Tamang B., Schillinger U., Franz C.M.A.P., Gores M., Holzapfe W.H. (2005). Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas. Int. J. Food Microbiol., 105: 347–356.

    Article  CAS  PubMed  Google Scholar 

  • Tannock G.W., Tilsala-Timisjärvi A., Rodtong S., Ng J., Munro K., Alatossava T. (1999). Identification ofLactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S–23S rRNA gene intergenic spacer region sequence comparisons. Appl. Environ. Microbiol., 65: 4264–4267.

    CAS  PubMed  Google Scholar 

  • Thompson J.D., Higgins D.G., Gibson T.J. (1994). CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acid. Res., 22: 4673–4680.

    Article  CAS  Google Scholar 

  • Tilsala-Timisjärvi A., Alatossava T. (1997). Development of oligonucleotide primers from 16S–23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Int. J. Food Microbiol., 35: 49–56.

    Article  PubMed  Google Scholar 

  • Woese C.R. (1987). Bacteria evolution. Microbiol. Rev., 51: 221–271.

    CAS  PubMed  Google Scholar 

  • Wong C.M.V.L., Sieo C.C., Low C.H., Abdullah N., Ho Y.W. (2005). Identification ofLactobacillus strains by specific amplification of 16S ribosomal DNA. The 8th Appl. Biol. Symp., Putrajaya, Malaysia, pp. 115.

  • Yin Q.Q., Zheng Q.H. (2005). Isolation and identification of the dominantLactobacillus in gut and faeces of pigs using carbohydrate fermentation and 16S rDNA analysis. J. Biosci. Bioeng., 99: 68–71.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Microbial Biotechnology Unit, Laboratory of Industrial Biotechnology, Institute of Bioscience, 43400 UPM Serdang, Selangor, Malaysia

    Chin Mei Lee, Chin Chin Sieo, Norhani Abdullah & Yin Wan Ho

  2. Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

    Chin Chin Sieo & Norhani Abdullah

  3. Biotechnology Research Institute, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia

    Clemente Michael Vui Ling Wong

Authors
  1. Chin Mei Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chin Chin Sieo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Clemente Michael Vui Ling Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Norhani Abdullah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yin Wan Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chin Chin Sieo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, C.M., Sieo, C.C., Wong, C.M.V.L. et al. Sequence analysis of 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region for differentiation of probioticsLactobacillus strains isolated from the gastrointestinal tract of chicken. Ann. Microbiol. 58, 133–140 (2008). https://doi.org/10.1007/BF03179457

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03179457

Key words

Search

Navigation