Skip to main content
SpringerLink
Log in

Evaluation of the Diversity of Probiotic Bacillus, Clostridium, and Bifidobacterium Using the Illumina-Based Sequencing Method

  • Published:
Probiotics and Antimicrobial Proteins Aims and scope Submit manuscript

Abstract

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.

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

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Colombo M, Castilho NPA, Todorov SD, Nero LA (2017) Beneficial and safety properties of a Corynebacteriun vitaeruminis strain isolated from cow rumen. Probiotics Antimicro Prot 9:102–110. https://doi.org/10.1007/s12602-017-9263-0

    Article  CAS  Google Scholar 

  2. Gaggia F, Mattarelli P, Biavati B (2010) Probiotics and prebiotics in animal feeding for safe food production. Int J Food Microbiol 141:S15–S28. https://doi.org/10.1016/j.ijfoodmicro.2010.02.031

    Article  PubMed  Google Scholar 

  3. Prasad J, Gill H, Smart J, Gopal PK (1998) Selection and characterization of Lactobacillus and Bifidobacterium dtrains for use as probiotics. Int Dairy J 8:993–1002. https://doi.org/10.1016/S0958-6946(99)00024-2

    Article  Google Scholar 

  4. Gomes AMP, Malcata FX (1999) Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends Food Sci Tech 10:139–157. https://doi.org/10.1016/S0924-2244(99)00033-3

    Article  CAS  Google Scholar 

  5. Cutting SM (2011) Bacillus probiotics. Food Microbiol 28:214–220. https://doi.org/10.1016/j.fm.2010.03.007

    Article  Google Scholar 

  6. Amit-Romach E, Sklan D, Uni Z (2004) Microflora ecology of the chicken intestine using 16S ribosomal DNA primers. Poultry Sci 83:1093–1098. https://doi.org/10.1093/ps/83.7.1093

    Article  CAS  Google Scholar 

  7. Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, Kjøller R, Kõljalg U, Pennanen T, Rosendahl S, Stenlid J, Kauserud H (2013) Fungal community analysis by high-throughput sequencing of amplified markers—a user’s guide. New Phytol 199:288–299. https://doi.org/10.1111/nph.12243

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Handl S, Dowd SE, Garcia-Mazcorro JF, Steiner JM, Suchodolski JS (2011) Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats. FEMS Microbiol Ecol 76:301–310. https://doi.org/10.1111/j.1574-6941.2011.01058.x

    Article  CAS  PubMed  Google Scholar 

  9. Hong S, Bunge J, Leslin C, Jeon S, Epstein SS (2009) Polymerase chain reaction primers miss half of rRNA microbial diversity. ISME J 3:1365–1373. https://doi.org/10.1038/ismej.2009.89

    Article  CAS  PubMed  Google Scholar 

  10. Maukonen J, Simões C, Saarela M (2012) The currently used commercial DNA-extraction methods gives different results of clostridial and actinobacterial populations derived from human fecal samples. FEMS Microbiol Ecol 79:697–708. https://doi.org/10.1111/j.1574-6941.2011.01257.x

    Article  CAS  PubMed  Google Scholar 

  11. Hill P, Kriśtůfek V, Dijkhuizen L, Boddy C, Kroetsch D, van Elsas JD (2011) Land use intensity controls actinobacterial community structure. Microb Ecol 61:286–302. https://doi.org/10.1007/s00248-010-9752-0

    Article  PubMed  Google Scholar 

  12. Duncan KR, Haltli B, Gill KA, Correa H, Berrué F, Kerr RG (2015) Exploring the diversity and metabolic potential of actinomycetes from temperate marine sediments from Newfoundland, Canada. J Ind Microbiol Biot 42:57–72. https://doi.org/10.1007/s10295-014-1529-x

    Article  CAS  Google Scholar 

  13. Du X, Zhai Y, Deng Q, Tan H, Cao L (2017) Rational selection for core actinobacterial endophytes in banana plants to promote growth of plantlets under Fusarium oxysporum f. sp. cubense (FOC) infested soils. Probiotics Antimicro Prot. doi:https://doi.org/10.1007/s12602-017-9293-7

    Article  Google Scholar 

  14. Wang W, Zhai Y, Cao L, Tan H, Zhang R (2016a) Illumina-based analysis of core actinobacteriome in roots, stems, and grains of rice. Microbiol Res 190:12–18. https://doi.org/10.1016/j.micres.2016.05.003

    Article  PubMed  Google Scholar 

  15. Wang W, Zhai Y, Cao L, Tan H, Zhang R (2016b) Endophytic bacterial and fungal microbiota in sprouts, roots and stems of rice (Oryza sativa L.) Microbiol Res 188-189:1–8. https://doi.org/10.1016/j.micres.2016.04.009

    Article  PubMed  Google Scholar 

  16. Kemp PF, Aller JY (2004) Bacterial diversity in aquatic and other environments: what 16S rDNA libraries can tell us. FEMS Microbiol Ecol 47:161–177. https://doi.org/10.1016/S0168-6496(03)00257-5

    Article  CAS  PubMed  Google Scholar 

  17. Kõljalg U, Nilsson RH, Abarenkov K et al (2013) Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol 22:5271–5277. https://doi.org/10.1111/mec.12481

    Article  CAS  PubMed  Google Scholar 

  18. Ondov BD, Bergman NH, Phillippy AM (2011) Interactive metagenomic visualization in a Web browser. BMC Bioinformatics 12:385. https://doi.org/10.1186/1471-2105-12-385

    Article  PubMed  PubMed Central  Google Scholar 

  19. Großkopf T, Soyer OS (2014) Synthetic microbial communities. Curr Opin Microbiol 18:72–77. https://doi.org/10.1016/j.mib.2014.02.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Oelschlaeger TA (2010) Mechanisms of probiotic actions—a review. Int J Med Microbiol 300:57–62. https://doi.org/10.1016/j.ijmm.2009.08.005

    Article  CAS  PubMed  Google Scholar 

  21. Wu XY, Walker MJ, Hornitzky M, Chin J (2006) Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance. J Microbiol Meth 64:107–119. https://doi.org/10.1016/j.mimet.2005.04.021

    Article  CAS  Google Scholar 

  22. Turroni F, Foroni E, Pizzetti P, Giubellini V, Ribbera A, Merusi P, Cagnasso P, Bizzarri B, de’Angelis GL, Shanahan F, Sinderen D, Ventura M (2009) Exploring the diversity of the bifidobacterial population in the human intestinal tract. Appl Environ Microbiol 75:1534–1545. https://doi.org/10.1128/AEM.02216-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bourhis AG, Saunier K, Dore J, Carlier JP, Chamba JF, Popoff MR, Tholozan JL (2005) Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradiet gel electrophoresis. Appl Environ Microbiol 71:29–38. https://doi.org/10.1128/AEM.71.1.29-38.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pan X, Wu T, Zhang L, Song Z, Tang H, Zhao Z (2008) In vitro evaluation on adherence and antimicrobial properties of a candidate probiotic Clostridium butyricum CB2 for farmed fish. J Appl Microbiol 105:1623–1629. https://doi.org/10.1111/j.1365-2672.2008.03885.x

    Article  CAS  PubMed  Google Scholar 

  25. Matsuki T, Watanabe K, Tanaka R, Fukuda M, Oyaizu H (1999) Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers. Appl Environ Microbiol 65:4506–4512

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Matsuki T, Watanabe K, Fujimoto J, Kado Y, Takada T, Matsumoto K, Tanaka R (2004) Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacetria. Appl Environ Microbiol 70:167–173. https://doi.org/10.1128/aem.70.1.167-173.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dyke MI, McCarthy AJ (2002) Molecular biological detection and characterization of Clostridium populations in municipal landfill sites. Appl Environ Microbiol 68:2049–2053. https://doi.org/10.1128/aem.68.4.2049-2053.2002

    Article  PubMed  PubMed Central  Google Scholar 

  28. Huang CH, Cheng CH, Cheng LH, Liang CM, Lin CY (2008) Application of Clostridium-specific PCR primers on the analysis of dark fermentation hydrogen-producing bactreial community. Int J Hydrogen Energ 33:1586–1592. https://doi.org/10.1016/j.ijhydene.2007.09.037

    Article  CAS  Google Scholar 

  29. Hirsch PR, Mauchline TH (2012) Who’s who in the plant root microbiome? Nat Biotechnol 30:961–962. https://doi.org/10.1117/12.203593

    Article  CAS  PubMed  Google Scholar 

  30. Jebaraj CS, Raghukumar C, Behnke A, Stoeck T (2010) Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation. FEMS Microbiol Ecol 71:399–412. https://doi.org/10.1111/j.1574-6941.2009.00804.x

    Article  CAS  PubMed  Google Scholar 

  31. Du X, Zhai Y, Deng Q, Tan H, Cao L (2017) Illumina-based sequencing analysis directed selection for Actinobacterial probiotic candidates for Banana plants. Probiotics Antimicrob Proteins. https://doi.org/10.1007/s12602-017-9293-7

    Article  Google Scholar 

Download references

Funding

This work was partly supported by grants from the Chinese National Natural Science Foundation (No. 31400111).

Author information

Authors and Affiliations

  1. School of Basic Courses, Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, China

    Zaichao Xu, Jun Liu & Zujun Deng

  2. School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Lixiang Cao & Hongming Tan

Authors
  1. Zaichao Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lixiang Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongming Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zujun Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zujun Deng.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, Z., Cao, L., Liu, J. et al. Evaluation of the Diversity of Probiotic Bacillus, Clostridium, and Bifidobacterium Using the Illumina-Based Sequencing Method. Probiotics & Antimicro. Prot. 10, 748–754 (2018). https://doi.org/10.1007/s12602-017-9337-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-017-9337-z

Keywords

Search

Navigation