title: Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling.
abstract: BACKGROUND
  The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling.
  
  
  RESULTS
  Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products.
  
  
  CONCLUSIONS
  Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies.
authors.affiliation: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av, Antônio Carlos 6627, 31,270-901, Belo Horizonte, MG, Brazil. moreira@icb.ufmg.br
authors.forename: João Luiz S
authors.initials: JL
authors.lastname: Moreira
authors.forename: Rodrigo M
authors.initials: RM
authors.lastname: Mota
authors.forename: Maria F
authors.initials: MF
authors.lastname: Horta
authors.forename: Santuza M R
authors.initials: SM
authors.lastname: Teixeira
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Neumann
authors.forename: Jacques R
authors.initials: JR
authors.lastname: Nicoli
authors.forename: Alvaro C
authors.initials: AC
authors.lastname: Nunes
mesh_terms: D000818:Animals; D002645:Chickens; D005516:Food Microbiology; D041981:Gastrointestinal Tract / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007778:Lactobacillus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D019936:Probiotics; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S