Abstract
Occurrence of bifidobacteria, known as health-promoting probiotic microorganisms, in the digestive tract of wild pigs (Sus scrofa) has not been examined yet. One hundred forty-nine fructose-6-phosphate phosphoketolase positive bacterial strains were isolated from colonic content of twenty-two individuals of wild pigs originated from four localities in the Czechia. Based on PCR-DGGE technique targeting the variable V3 region of the 16S rRNA genes, strains were initially differentiated into four groups represented by: (i) probably a new Bifidobacterium species (89 strains), (ii) B. boum/B. thermophilum/B. thermacidophilum subsp. porcinum/B. thermacidophilum subsp. thermacidophilum (sub)species (49 strains), (iii) Pseudoscardovia suis (7 strains), and (iv) B. pseudolongum subsp. globosum/B. pseudolongum subsp. pseudolongum (4 strains), respectively. Given the fact that DGGE technique did not allow to differentiate the representatives of thermophilic bifidobacteria and B. pseudolongum subspecies, strains were further classified by the 16S rRNA and thrS gene sequences. Primers targeting the variable regions of the latter gene were designed to be applicable in identification and phylogeny of Bifidobacteriaceae family. The 16S rRNA-derived phylogenetic study classified members of the first group into five subgroups in a separated cluster of thermophilic bifidobacteria. Comparable results were obtained by the thrS-derived phylogenetic analysis. Remarkably, variability among thrS sequences was higher compared with 16S rRNA gene sequences. Overall, molecular genetic techniques application allowed to identify a new Bifidobacterium phylotype which is predominant in the digestive tract of examined wild pigs.
Similar content being viewed by others
References
Biavati B, Vescovo M, Torriani S, Bottazzi V (2000) Bifidobacteria: history, ecology, physiology and applications. Ann Microbiol 50:117–131
Bunesova V, Killer J, Javurkova B, Vlkova E, Tejnecky V, Musilova S, Rada V (2017) Diversity of the subspecies Bifidobacterium animalis subsp. lactis. Anaerobe 44:40–47
Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552
Crociani F, Alessandrini A, Mucci MM, Biavati B (1994) Degradation of complex carbohydrates by Bifidobacterium spp. Int J Food Microbiol 24:199–210
Delorme C, Poyart C, Ehrlich SD, Renault P (2007) Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group. J Bacteriol 189:1330–1341
Endo A, Futagawa-Endo Y, Schumann P, Pukall R, Dicks LM (2012) 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) and red-handed tamarin (Saguinus midas). Syst Appl Microbiol 35:92–97
Gavini F, Delcenserie V, Kopeinig K, Pollinger S, Beerens H, Bonaparte C, Upmann M (2006) Bifidobacterium species isolated from animal feces and from beef and pork meat. J Food Prot 69:871–877
Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW (2000) Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30:61–67
Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Nageshwar Reddy D (2015) Role of the normal gut microbiota. World J Gastroenterol 21:8787–8803
Jans C, de Wouters T, Bonfoh B, Lacroix C, Kaindi DW, Anderegg J, Böck D, Vitali S, Schmid T, Isenring J, Kurt F, Kogi-Makau W, Meile L (2016) Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme. BMC Microbiol 16:117
Killer J, Kopečný J, Mrázek J, Rada V, Dubná S, Marounek M (2010) Bifidobacteria in the digestive tract of bumblebees. Anaerobe 16:165–170
Killer J, Mrázek J, Bunešová V, Havlík J, Koppová I, Benada O, Rada V, Kopečný J, Vlková E (2013) 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 36:11–16
Killer J, Ročková Š, Vlková E, Rada V, Havlík J, Kopečný J, Bunesová V, Benada O, Kofronová O, Pechar R, Profousová I (2013) 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 63:4439–4446
Killer J, Havlik J, Bunesova V, Vlkova E, Benada O (2014) Pseudoscardovia radai sp. nov., another representative of a new genus within the family Bifidobacteriaceae isolated from the digestive tract of a wild pig (Sus scrofa scrofa). Int J Syst Evol Microbiol 64:2932–2938
Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721
Kim M, Oh HS, Park SC, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346–351
Lamendella R, Santo Domingo JW, Kelty C, Oerther DB (2008) Bifidobacteria in feces and environmental waters. Appl Environ Microbiol 74:575–584
Maxwell FJ, Duncan SH, Hold G, Stewart CS (2004) Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs. Anaerobe 10:33–39
Mikkelsen LL, Bendixen C, Jakobsen M, Jensen BB (2003) Enumeration of bifidobacteria in gastrointestinal samples from piglets. Appl Environ Microbiol 69:654–658
Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ekology. Antonie Van Leeuwenhoek 73:127–141
Ochman H, Worobey M, Kuo CH, Ndjango JB, Peeters M, Hahn BH, Hugenholtz P (2010) Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol 8(11):e1000546
Orban JI, Patterson JA (2000) Modification of the phosphoketolase assay for rapid identification of bifidobacteria. J Microbiol Methods 40:221–224
Rada V, Petr J (2000) A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca. J Microbiol Methods 43:127–132
Rada V, Petr J (2002) Enumeration of bifidobacteria in animal intestinal samples. Vet Med Czech 47:1–4
Scardovi V, Trovatelli LD, Biavati B, Zani G (1979) Bifidobacterium cuniculi, Bifidobacterium choerinum, Bifidobacterium boum, and Bifidobacterium pseudocatenulatum: four new species and their deoxyribonucleic acid homology relationships. Int J Syst Bacteriol 29:291–311
Shkoporov AN, Khokhlova EV, Kulagina EV, Smeianov VV, Kafarskaia LI, Efimov BA (2008) Application of several molecular techniques to study numerically predominant Bifidobacterium spp. and Bacteroidales order strains in the feces of healthy children. Biosci Biotechnol Biochem 72:742–748
Silvi S, Verdenelli MC, Orpianesi C, Cresci A (2003) EU project Crownalife: functional foods, gut microflora and healthy ageing. Isolation and identification of Lactobacillus and Bifidobacterium strains from faecal samples of elderly subjects for a possible probiotic use in functional foods. J Food Eng 56:195–200
Simpson JM, McCracken VJ, Gaskins HR, Mackie RI (2000) Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl Environ Microbiol 66:4705–4714
Simpson PJ, Stanton C, Fitzgerald GF, Ross RP (2003) Genomic diversity and relatedness of bifidobacteria isolated from a porcine cecum. J Bacteriol 185:2571–2581
Simpson PJ, Ross RP, Fitzgerald GF, Stanton C (2004) Bifidobacterium psychraerophilum sp. nov. and Aeriscardovia aeriphila gen. nov., sp. nov., isolated from a porcine caecum. Int J Syst Evol Microbiol 54:401–406
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729
Trebichavsky I, Rada V, Splichalova A, Splichal I (2009) Cross-talk of human gut with bifidobacteria. Nutr Rev 67:77–82
Vlková E, Trojanová I, Rada V (2006) Distribution of bifidobacteria in the gastrointestinal tract of calves. Folia Microbiol 51:325–328
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 7:79
Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP (2015) 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome 3:26
Yanokura E, Oki K, Makino H, Modesto M, Pot B, Mattarelli P, Biavati B, Watanabe K (2015) Subspeciation of Bifidobacterium longum by multilocus approaches and amplified fragment length polymorphism: description of B. longum subsp. suillum subsp. nov., isolated from the faeces of piglets. Syst Appl Microbiol 38:305–314
Zhang J, Kong Y, Feng Y, Huang J, Song T, Ruan Z, Song J, Jiang Y, Yu Y, Xie X (2014) Development of a multilocus sequence typing scheme for Ureaplasma. Eur J Clin Microbiol Infect Dis 33:537–544
Zhu L, Li W, Dong X (2003) Species identification of genus Bifidobacterium based on partial HSP60 gene sequences and proposal of Bifidobacterium thermacidophilum subsp. porcinum subsp. nov. Int J Syst Evol Microbiol 53:1619–1623
Acknowledgements
This work was supported by the by the Grant Agency of the Czech Republic (Project No. GA13-08803S).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflicts of interest
The authors declare that they have no potential conflict of interest related to this study.
Ethical Approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in the study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Pechar, R., Killer, J., Mekadim, C. et al. Classification of Culturable Bifidobacterial Population from Colonic Samples of Wild Pigs (Sus scrofa) Based on Three Molecular Genetic Methods. Curr Microbiol 74, 1324–1331 (2017). https://doi.org/10.1007/s00284-017-1320-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-017-1320-0