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Volume 163, Issue 7

Effective identification of group species: genome-based selection of the gene as the target of a novel multiplex PCR assay Free

Abstract

and form a closely related taxonomic group (the group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish , and strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): identification , L. casei group species , multiplex PCR and mutL
© 2017 The Authors
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2017-07-01
2024-04-20
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References

  1. Ashraf R, Shah NP. Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt–a review. Int J Food Microbiol 2011; 149:194–208 [View Article][PubMed]
     [Google Scholar]
  2. Felis GE, Dellaglio F. Taxonomy of lactobacilli and bifidobacteria. Curr Issues Intest Microbiol 2007; 8:44–61[PubMed]
     [Google Scholar]
  3. Salvetti E, Torriani S, Felis GE. The genus Lactobacillus: a taxonomic update. Probiotics Antimicrob Proteins 2012; 4:217–226 [View Article][PubMed]
     [Google Scholar]
  4. Liévin-Le Moal V, Servin AL. Anti-infective activities of Lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clin Microbiol Rev 2014; 27:167–199 [View Article][PubMed]
     [Google Scholar]
  5. Matsuzaki T. Immunomodulation by treatment with Lactobacillus casei strain Shirota. Int J Food Microbiol 1998; 41:133–140 [View Article][PubMed]
     [Google Scholar]
  6. Cosenza L, Nocerino R, di Scala C, di Costanzo M, Amoroso A et al. Bugs for atopy: the Lactobacillus rhamnosus GG strategy for food allergy prevention and treatment in children. Benef Microbes 2015; 6:225–232 [View Article][PubMed]
     [Google Scholar]
  7. Ouwehand A, Isolauri E, Salminen S. The role of the intestinal microflora for the development of the immune system in early childhood. Eur J Nutr 2002; 41:132–137 [View Article][PubMed]
     [Google Scholar]
  8. Desai AR, Shah NP, Powell IB. Discrimination of dairy industry isolates of the Lactobacillus casei group. J Dairy Sci 2006; 89:3345–3351 [View Article][PubMed]
     [Google Scholar]
  9. Toh H, Oshima K, Nakano A, Takahata M, Murakami M et al. Genomic adaptation of the Lactobacillus casei group. PLoS One 2013; 8:e75073 [View Article][PubMed]
     [Google Scholar]
  10. Gatti M, Bottari B, Lazzi C, Neviani E, Mucchetti G. Invited review: microbial evolution in raw-milk, long-ripened cheeses produced using undefined natural whey starters. J Dairy Sci 2014; 97:573–591 [View Article][PubMed]
     [Google Scholar]
  11. Dellaglio F, Felis GE, Torriani S. The status of the species Lactobacillus casei (Orla-Jensen 1916) Hansen and Lessel 1971 and Lactobacillus paracasei Collins et al. 1989. Request for an opinion. Int J Syst Evol Microbiol 2002; 52:285–287 [View Article][PubMed]
     [Google Scholar]
  12. Dicks LM, du Plessis EM, Dellaglio F, Lauer E. Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosus ATCC 15820 as Lactobacillus zeae nom. rev., designation of ATCC 334 as the neotype of L. casei subsp. casei, and rejection of the name Lactobacillus paracasei. Int J Syst Bacteriol 1996; 46:337–340 [View Article][PubMed]
     [Google Scholar]
  13. Wayne LG. Actions of the judicial commission of the International Committee on Systematic Bacteriology on requests for opinions published between January 1985 and July 1993. Int J Syst Bacteriol 1994:177–178
     [Google Scholar]
  14. Tindall BJ. Judicial Commission of the International Committee on Systematics of Bacteria The type strain of Lactobacillus casei is ATCC 393, ATCC 334 cannot serve as the type because it represents a different taxon, the name Lactobacillus paracasei and its subspecies names are not rejected and the revival of the name 'Lactobacillus zeae' contravenes Rules 51b (1) and (2) of the International Code of Nomenclature of Bacteria. Opinion 82. Int J Syst Evol Microbiol 2008; 58:1764–1765 [View Article][PubMed]
     [Google Scholar]
  15. Sun Z, Harris HM, Mccann A, Guo C, Argimón S et al. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat Commun 2015; 6:8322 [View Article][PubMed]
     [Google Scholar]
  16. Huang C-H, Chang M-T, Huang L. Use of highly variable gene (yycH) as DNA marker to resolve interspecific relationships within the Lactobacillus casei group and a target for developing novel species-specific PCR primers. Eur Food Res Technol 2014; 239:719–724 [View Article]
     [Google Scholar]
  17. Iacumin L, Ginaldi F, Manzano M, Anastasi V, Reale A et al. High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR. Food Microbiol 2015; 46:357–367 [View Article][PubMed]
     [Google Scholar]
  18. Karimi R, Mortazavian AM, Amiri-Rigi A. Selective enumeration of probiotic microorganisms in cheese. Food Microbiol 2012; 29:1–9 [View Article][PubMed]
     [Google Scholar]
  19. Vásquez A, Ahrné S, Pettersson B, Molin G. Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus. Lett Appl Microbiol 2001; 32:215–219 [View Article][PubMed]
     [Google Scholar]
  20. Vásquez A, Molin G, Pettersson B, Antonsson M, Ahrné S. DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species. Syst Appl Microbiol 2005; 28:430–441 [View Article][PubMed]
     [Google Scholar]
  21. Colombo M, de Oliveira AE, de Carvalho AF, Nero LA. Development of an alternative culture medium for the selective enumeration of Lactobacillus casei in fermented milk. Food Microbiol 2014; 39:89–95 [View Article][PubMed]
     [Google Scholar]
  22. Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R. Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 2003; 69:6908–6922 [View Article][PubMed]
     [Google Scholar]
  23. Felis GE, Dellaglio F, Mizzi L, Torriani S. Comparative sequence analysis of a recA gene fragment brings new evidence for a change in the taxonomy of the Lactobacillus casei group. Int J Syst Evol Microbiol 2001; 51:2113–2117 [View Article][PubMed]
     [Google Scholar]
  24. Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K et al. Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 2007; 57:2777–2789 [View Article][PubMed]
     [Google Scholar]
  25. Watanabe K, Fujimoto J, Sasamoto M, Dugersuren J, Tumursuh T et al. Diversity of lactic acid bacteria and yeasts in Airag and Tarag, traditional fermented milk products of Mongolia. World J Microbiol Biotechnol 2008; 24:1313–1325 [View Article]
     [Google Scholar]
  26. Huang CH, Lee FL. The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group. Antonie van Leeuwenhoek 2011; 99:319–327 [View Article][PubMed]
     [Google Scholar]
  27. Huang CH, Chang MT, Huang MC, Lee FL. Application of the SNaPshot minisequencing assay to species identification in the Lactobacillus casei group. Mol Cell Probes 2011; 25:153–157 [View Article][PubMed]
     [Google Scholar]
  28. Adimpong DB, Nielsen DS, Sørensen KI, Vogensen FK, Sawadogo-Lingani H et al. Lactobacillus delbrueckii subsp. jakobsenii subsp. nov., isolated from dolo wort, an alcoholic fermented beverage in Burkina Faso. Int J Syst Evol Microbiol 2013; 63:3720–3726 [View Article][PubMed]
     [Google Scholar]
  29. Cai H, Rodríguez BT, Zhang W, Broadbent JR, Steele JL. Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specificity. Microbiology 2007; 153:2655–2665 [View Article][PubMed]
     [Google Scholar]
  30. Chaillou S, Lucquin I, Najjari A, Zagorec M, Champomier-Vergès MC. Population genetics of Lactobacillus sakei reveals three lineages with distinct evolutionary histories. PLoS One 2013; 8:e73253 [View Article][PubMed]
     [Google Scholar]
  31. Dan T, Liu W, Song Y, Xu H, Menghe B et al. Erratum: the evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST). BMC Microbiol 2016; 16:52 [View Article][PubMed]
     [Google Scholar]
  32. de Las Rivas B, Marcobal A, Muñoz R. Development of a multilocus sequence typing method for analysis of Lactobacillus plantarum strains. Microbiology 2006; 152:85–93 [View Article][PubMed]
     [Google Scholar]
  33. Diancourt L, Passet V, Chervaux C, Garault P, Smokvina T et al. Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination. Appl Environ Microbiol 2007; 73:6601–6611 [View Article][PubMed]
     [Google Scholar]
  34. Gosiewski T, Brzychczy-Włoch M, Heczko PB. The application of multiplex PCR to detect seven different DNA targets in group B streptococci. Folia Microbiol 2012; 57:163–167 [View Article][PubMed]
     [Google Scholar]
  35. O' Donnell MM, Harris HM, Lynch DB, Ross RP, O'Toole PW. Lactobacillus ruminis strains cluster according to their mammalian gut source. BMC Microbiol 2015; 15:80 [View Article][PubMed]
     [Google Scholar]
  36. Parolo CC, do T, Henssge U, Alves LS, de Santana Giongo FC et al. Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms. J Appl Microbiol 2011; 111:105–113 [View Article][PubMed]
     [Google Scholar]
  37. Picozzi C, Bonacina G, Vigentini I, Foschino R. Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses. Microbiology 2010; 156:2035–2045 [View Article][PubMed]
     [Google Scholar]
  38. Raftis EJ, Salvetti E, Torriani S, Felis GE, O'Toole PW. Genomic diversity of Lactobacillus salivarius. Appl Environ Microbiol 2011; 77:954–965 [View Article][PubMed]
     [Google Scholar]
  39. Ramachandran P, Lacher DW, Pfeiler EA, Elkins CA. Development of a tiered multilocus sequence typing scheme for members of the Lactobacillus acidophilus complex. Appl Environ Microbiol 2013; 79:7220–7228 [View Article][PubMed]
     [Google Scholar]
  40. Su MS, Oh PL, Walter J, Gänzle MG. Intestinal origin of sourdough Lactobacillus reuteri isolates as revealed by phylogenetic, genetic, and physiological analysis. Appl Environ Microbiol 2012; 78:6777–6780 [View Article][PubMed]
     [Google Scholar]
  41. Tanganurat W, Quinquis B, Leelawatcharamas V, Bolotin A. Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables. J Basic Microbiol 2009; 49:377–385 [View Article][PubMed]
     [Google Scholar]
  42. Woese CR. Bacterial evolution. Microbiol Rev 1987; 51:221–271[PubMed]
     [Google Scholar]
  43. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797 [View Article][PubMed]
     [Google Scholar]
  44. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013; 14:60 [View Article][PubMed]
     [Google Scholar]
  45. Auch AF, von Jan M, Klenk HP, Göker M. Digital DNA–DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2010; 2:117–134 [View Article][PubMed]
     [Google Scholar]
  46. Ward LJ, Timmins MJ. Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett Appl Microbiol 1999; 29:90–92 [View Article][PubMed]
     [Google Scholar]
  47. Mora D, Fortina MG, Parini C, Daffonchio D, Manachini PL. Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains. Microbiology 2000; 146:2027–2038 [View Article][PubMed]
     [Google Scholar]
  48. de Las Rivas B, Marcobal A, Muñoz R. Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes. Appl Environ Microbiol 2004; 70:7210–7219 [View Article][PubMed]
     [Google Scholar]
  49. Diancourt L, Passet V, Chervaux C, Garault P, Smokvina T et al. Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination. Appl Environ Microbiol 2007; 73:6601–6611 [View Article][PubMed]
     [Google Scholar]
  50. Bilhère E, Lucas PM, Claisse O, Lonvaud-Funel A. Multilocus sequence typing of Oenococcus oeni: detection of two subpopulations shaped by intergenic recombination. Appl Environ Microbiol 2009; 75:1291–1300 [View Article][PubMed]
     [Google Scholar]
  51. Martino ME, Maifreni M, Marino M, Bartolomeoli I, Carraro L et al. Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon. Antonie van Leeuwenhoek 2013; 103:1149–1163 [View Article][PubMed]
     [Google Scholar]
  52. Dan T, Liu W, Sun Z, Lv Q, Xu H et al. A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia. BMC Microbiol 2014; 14:150 [View Article][PubMed]
     [Google Scholar]
  53. Rahkila R, Johansson P, Säde E, Paulin L, Auvinen P et al. Multilocus sequence typing of Leuconostoc gelidum subsp. gasicomitatum, a psychrotrophic lactic acid bacterium causing spoilage of packaged perishable foods. Appl Environ Microbiol 2015; 81:2474–2480 [View Article][PubMed]
     [Google Scholar]
  54. Bove CG, de Angelis M, Gatti M, Calasso M, Neviani E et al. Metabolic and proteomic adaptation of Lactobacillus rhamnosus strains during growth under cheese-like environmental conditions compared to de Man, Rogosa, and Sharpe medium. Proteomics 2012; 12:3206–3218 [View Article][PubMed]
     [Google Scholar]
  55. Pogačić T, Mancini A, Santarelli M, Bottari B, Lazzi C et al. Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter. Food Microbiol 2013; 36:207–215 [View Article][PubMed]
     [Google Scholar]
  56. Lazzi C, Turroni S, Mancini A, Sgarbi E, Neviani E et al. Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese. BMC Microbiol 2014; 7:14–28 [View Article][PubMed]
     [Google Scholar]
  57. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014; 11:506–514 [View Article][PubMed]
     [Google Scholar]
  58. Mori K, Yamazaki K, Ishiyama T, Katsumata M, Kobayashi K et al. Comparative sequence analyses of the genes coding for 16S rRNA of Lactobacillus casei-related taxa. Int J Syst Bacteriol 1997; 47:54–57 [View Article][PubMed]
     [Google Scholar]
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Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay
Microbiology 163, 950 (2017); https://doi.org/10.1099/mic.0.000497
/content/journal/micro/10.1099/mic.0.000497
/content/journal/micro/10.1099/mic.0.000497
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