Identification of Species Belonging to the Bifidobacterium Genus by PCR-RFLP Analysis of a Hsp60 Gene Fragment

Overview

Journal BMC Microbiol

Publisher Biomed Central

Specialty Microbiology

Date 2013 Jul 3

PMID 23815602

Citations 5

Authors

Loredana Baffoni

Verena Stenico

Erwin Strahsburger

Francesca Gaggia

Diana Di Gioia

Monica Modesto

Paola Mattarelli

Bruno Biavati

Affiliations

Soon will be listed here.

Abstract

Background: Bifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.

Results: This work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590 bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns.

Conclusions: The developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.

Citing Articles

Benefits and limitations of a new genome-based PCR-RFLP genotyping assay (GB-RFLP): A SNP-based detection method for identification of species in extremely young adaptive radiations.

Kratochwil C, Kautt A, Rometsch S, Meyer A Ecol Evol. 2022; 12(3):e8751.

PMID: 35356554 PMC: 8941502. DOI: 10.1002/ece3.8751.

Identification, Characterization, and Antioxidant Potential of subsp. Strains Isolated From Feces of Healthy Infants.

Zhao L, Wang S, Dong J, Shi J, Guan J, Liu D Front Microbiol. 2021; 12:756519.

PMID: 34795651 PMC: 8593421. DOI: 10.3389/fmicb.2021.756519.

In Vitro Comparative Analysis of Probiotic and Functional Attributes of Indigenous Isolates of Bifidobacteria.

Achi S, Halami P Curr Microbiol. 2019; 76(3):304-311.

PMID: 30600360 DOI: 10.1007/s00284-018-1615-9.

Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products.

Barnaba T, Gangiredla J, Mammel M, Lacher D, Elkins C, Lampel K Genome Announc. 2018; 6(26).

PMID: 29954907 PMC: 6025948. DOI: 10.1128/genomeA.00610-18.

Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

Jarocki P, Podlesny M, Komon-Janczara E, Kucharska J, Glibowska A, Targonski Z BMC Microbiol. 2016; 16(1):159.

PMID: 27449060 PMC: 4957357. DOI: 10.1186/s12866-016-0779-3.

References

Vincent D, Roy D, Mondou F, Dery C . Characterization of bifidobacteria by random DNA amplification. Int J Food Microbiol. 1998; 43(3):185-93. DOI: 10.1016/s0168-1605(98)00109-3. View

Jian W, Zhu L, Dong X . New approach to phylogenetic analysis of the genus Bifidobacterium based on partial HSP60 gene sequences. Int J Syst Evol Microbiol. 2001; 51(Pt 5):1633-1638. DOI: 10.1099/00207713-51-5-1633. View

Rossi M, Altomare L, Gonzalez Vara y Rodriguez A, Brigidi P, Matteuzzi D . Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene. Arch Microbiol. 2000; 174(1-2):74-80. DOI: 10.1007/s002030000178. View

Matsuki T, Watanabe K, Tanaka R, Fukuda M, Oyaizu H . Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers. Appl Environ Microbiol. 1999; 65(10):4506-12. PMC: 91600. DOI: 10.1128/AEM.65.10.4506-4512.1999. View

Bikandi J, San Millan R, Rementeria A, Garaizar J . In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction. Bioinformatics. 2004; 20(5):798-9. DOI: 10.1093/bioinformatics/btg491. View

Hill J, Penny S, Crowell K, Goh S, Hemmingsen S . cpnDB: a chaperonin sequence database. Genome Res. 2004; 14(8):1669-75. PMC: 509277. DOI: 10.1101/gr.2649204. View

Krizova J, Spanova A, Rittich B . Evaluation of amplified ribosomal DNA restriction analysis (ARDRA) and species-specific PCR for identification of Bifidobacterium species. Syst Appl Microbiol. 2006; 29(1):36-44. DOI: 10.1016/j.syapm.2005.07.003. View

Matsuki T, Watanabe K, Tanaka R . Genus- and species-specific PCR primers for the detection and identification of bifidobacteria. Curr Issues Intest Microbiol. 2003; 4(2):61-9. View

Wong R, Chow A . Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences. FEMS Microbiol Lett. 2002; 206(1):107-13. DOI: 10.1111/j.1574-6968.2002.tb10994.x. View

10.

Morita H, Nakano A, Onoda H, Toh H, Oshima K, Takami H . Bifidobacterium kashiwanohense sp. nov., isolated from healthy infant faeces. Int J Syst Evol Microbiol. 2010; 61(Pt 11):2610-2615. DOI: 10.1099/ijs.0.024521-0. View

11.

Blaiotta G, Fusco V, Ercolini D, Aponte M, Pepe O, Villani F . Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation. Appl Environ Microbiol. 2007; 74(1):208-15. PMC: 2223197. DOI: 10.1128/AEM.01711-07. View

12.

Biavati B, Castagnoli P, Trovatelli L . Species of the genus Bifidobacterium in the feces of human adults. Microbiologica. 1986; 9(1):39-45. View

13.

Baffoni L, Gaggia F, Di Gioia D, Santini C, Mogna L, Biavati B . A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain. Int J Food Microbiol. 2012; 157(2):156-61. DOI: 10.1016/j.ijfoodmicro.2012.04.024. View

14.

Rusanganwa E, Singh B, Gupta R . Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach. Biochim Biophys Acta. 1992; 1130(1):90-4. DOI: 10.1016/0167-4781(92)90468-f. View

15.

Ventura M, Canchaya C, Del Casale A, Dellaglio F, Neviani E, Fitzgerald G . Analysis of bifidobacterial evolution using a multilocus approach. Int J Syst Evol Microbiol. 2006; 56(Pt 12):2783-2792. DOI: 10.1099/ijs.0.64233-0. View

16.

Matsuki T, Watanabe K, Tanaka R, Oyaizu H . Rapid identification of human intestinal bifidobacteria by 16S rRNA-targeted species- and group-specific primers. FEMS Microbiol Lett. 1998; 167(2):113-21. DOI: 10.1111/j.1574-6968.1998.tb13216.x. View

17.

Endo A, Futagawa-Endo Y, Schumann P, Pukall R, Dicks L . Bifidobacterium reuteri sp. nov., Bifidobacterium callitrichos sp. nov., Bifidobacterium saguini sp. nov., Bifidobacterium stellenboschense sp. nov. and Bifidobacterium biavatii sp. nov. isolated from faeces of common marmoset (Callithrix jacchus).... Syst Appl Microbiol. 2012; 35(2):92-7. DOI: 10.1016/j.syapm.2011.11.006. View

18.

Kim M, Roh S, Bae J . Bifidobacterium stercoris sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. 2010; 60(Pt 12):2823-2827. DOI: 10.1099/ijs.0.019943-0. View

19.

Matsuki T, Watanabe K, Fujimoto J, Kado Y, Takada T, Matsumoto K . Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria. Appl Environ Microbiol. 2004; 70(1):167-73. PMC: 321263. DOI: 10.1128/AEM.70.1.167-173.2004. View

20.

Ventura M, Meylan V, Zink R . Identification and tracing of Bifidobacterium species by use of enterobacterial repetitive intergenic consensus sequences. Appl Environ Microbiol. 2003; 69(7):4296-301. PMC: 165193. DOI: 10.1128/AEM.69.7.4296-4301.2003. View