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Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

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Abstract

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.

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References

  1. Beresford TP, Fitzsimons NA, Brennan NL, Cogan TM (2001) Recent advances in cheese microbiology. Int Dairy J 11:259–274

    Article  CAS  Google Scholar 

  2. Bottari B, Agrimonti C, Gatti M, Neviani E, Marmiroli N (2013) Development of a multiplex real-time PCR to detect thermophilic lactic acid bacteria in natural whey starters. Int J Food Microbiol 160:290–297

    Article  CAS  PubMed  Google Scholar 

  3. Bujnakova D, Kmet V (2012) Functional properties of Lactobacillus strains isolated from dairy products. Folia Microbiol 57:263–267

    Article  CAS  Google Scholar 

  4. Callanan M, Kaleta P, O’Callaghan J, O′Sullivan O, Jordan K, McAuliffe O, Sangrador-Vegas A, Slattery L, Fitzgerald GF, Beresford T, Ross RP (2008) Genome sequence of L. helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J Bacteriol 190:727–735

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Cremonesi P, Vanoni L, Morandi S, Silvetti T, Castiglioni B, Brasca M (2011) Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese. Int J Food Microbiol 146:207–211

    Article  CAS  PubMed  Google Scholar 

  6. De los Reyes-Gavilan CG, Limsowtin GKY, Tailliez P, Sechaud L, Accolas JP (1992) A L. helveticus—specific DNA probe detects restriction fragment length polymorphisms in this species. Appl Environ Microbiol 58:3429–3432

    CAS  PubMed Central  PubMed  Google Scholar 

  7. Du Plessis EM, Dicks LMT (1995) Evaluation of random amplified polymorphic DNA (RAPD)—PCR as a method to differentiate L. acidophilus, L. crispatus, L. amylovorus, L. gallinarum, L. gasseri, and L. johnsonii. Curr Microbiol 31:114–118

    Article  PubMed  Google Scholar 

  8. Felis GE, Dellaglio F (2007) Taxonomy of lactobacilli and bifidobacteria. Curr Issues Intest Microbiol 8:44–61

    CAS  PubMed  Google Scholar 

  9. Fortina MG, Ricci G, Mora D, Parini C, Manachini PL (2001) Specific identification of L. helveticus by PCR with pepC, pepN and htrA targeted primers. FEMS Microbiol Lett 198:85–89

    Article  CAS  PubMed  Google Scholar 

  10. Fujisawa T, Benno Y, Yaeshima T, Mitsuoka T (1992) Taxonomic study of the L. acidophilus group, with recognition of L.  gallinarum sp. nov. and L. johnsonii sp. nov. and synonymy of L. acidophilus Groupe A3 (Johnson et al. 1980) with the type strain of L. amylovorus (Nakamura et al. 1981). Int J Syst Bacteriol 42:487–491

    Article  CAS  PubMed  Google Scholar 

  11. Guan LL, Hagen KE, Tannock GW, Korver DR, Fasenko GM, Allison GE (2003) Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl Environ Microbiol 69:6750–6757

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Holzapfel WH, Wood BJB (1998) The genera of lactic acid bacteria. Blackie Academic and Professional, London

    Google Scholar 

  13. Hong GE, Kim DG, Bae JY, Ahn SH, Bai SC, Kong IS (2007) Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-l-alanine amidase. FEMS Microbiol Lett 269:201–206

    Article  CAS  PubMed  Google Scholar 

  14. Huang CH, Chang MT, Huang MC, Wang LT, Huang L, Lee FL (2012) Discrimination of the L. acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene. J Sci Food Agric 92:2703–2708

    Article  CAS  PubMed  Google Scholar 

  15. Jauhiainen T, Niittynen L, Oreisic M, Järvenpää S, Hiltunen TP, Rönnback M, Vapaatalo H, Korpela R (2012) Effects of long-term intake of lactotripeptides on cardiovascular risk factors in hypertensive subjects. Eur J Clin Nutr 66:843–849

    Article  CAS  PubMed  Google Scholar 

  16. Jebava I, Plockova M, Lortal S, Valence F (2011) The nine peptidoglycan hydrolases genes in L. helveticus are ubiquitous and early transcribed. Int J Food Microbiol 148:1–7

    Article  CAS  PubMed  Google Scholar 

  17. Kandler O, Weiss N (1986) Regular nonsporing gram-positive rods. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds) Bergey′s manual of systematic bacteriology, vol 2., Williams and Wilkins CoBaltimore, MD, pp 1208–1234

    Google Scholar 

  18. Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR (2000) Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the L. acidophilus complex. J Appl Microbiol 89:511–516

    Article  CAS  PubMed  Google Scholar 

  19. Lortal S, Valence F, Bizet C, Maubois JL (1997) Electrophoretic pattern of peptidoglycan hydrolases, a new tool for bacterial species identification: application to 10 Lactobacillus species. Res Microbiol 148:461–474

    Article  CAS  PubMed  Google Scholar 

  20. Meroth CB, Hammes WP, Hertel C (2004) Characterisation of the microbiota of rice sourdoughs and description of L. spicheri sp. nov. Syst Appl Microbiol 27:151–159

    Article  CAS  PubMed  Google Scholar 

  21. Moroni AV, Arendt EK, Bello FD (2011) Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs. Food Microbiol 28:497–502

    Article  PubMed  Google Scholar 

  22. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  23. Singh S, Goswami P, Singh R, Heller KJ (2009) Application of molecular identification tools for Lactobacillus, with a focus on discrimination between closely related species: a review. LWT Food Sci Technol 42:448–457

    Article  CAS  Google Scholar 

  24. Slattery L, O’Callaghan J, Fitzgerald GF, Beresford T, Ross RP (2010) Invited review: L. helveticus—a thermophilic dairy starter related to gut bacteria. J Dairy Sci 93:4435–4454

    Article  CAS  PubMed  Google Scholar 

  25. Stackebrandt E, Frederiksen W, Garrity GM, Grimont PAD, Kampfer P, Maiden MCJ, Nesme X, Rossello-Mora R, Swings J, Truper HG, Vauterin L, Ward AC, Whitman BW (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52:1043–1047

    Article  CAS  PubMed  Google Scholar 

  26. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed  Google Scholar 

  27. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74

    Article  PubMed Central  PubMed  Google Scholar 

  28. Valence F, Lortal S (1995) Zymogram and preliminary characterization of L. helveticus autolysins. Appl Environ Microbiol 61:3391–3399

    CAS  PubMed Central  PubMed  Google Scholar 

  29. Van Hoorde K, Verstraete T, Vandamme P, Huys G (2008) Diversity of lactic acid bacteria in two Flemish artisan raw milk Gouda-type cheeses. Food Microbiol 25:929–935

    Article  PubMed  Google Scholar 

  30. Ventura M, Callegari ML, Morelli L (2000) S-layer gene as a molecular marker for identification of L. helveticus. FEMS Microbiol Lett 189:275–279

    Article  CAS  PubMed  Google Scholar 

  31. Ventura M, Canchaya C, Meylan V, Klaenhammer TR, Zink R (2003) Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Appl Environ Microbiol 69:6908–6922

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Viiard E, Mihhalevski A, Rühka T, Paalme T, Sarand I (2013) Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed L. helveticus as the dominant species. J Appl Microbiol 114:404–412

    Article  CAS  PubMed  Google Scholar 

  33. Vinogradov E, Valence F, Maes E, Jebava I, Chuat V, Lortal S, Grard T, Guerardel Y, Sadovskaya I (2013) Structural studies of the cell wall polysaccharides from three strains of L. helveticus with different autolytic properties: DPC4571, BROI, and LH1. Carbohydr Res 379:7–12

    Article  CAS  PubMed  Google Scholar 

  34. Wine E, Gareau MG, Johnson-Henry K, Sherman PM (2009) Strain-specific probiotic L. helveticus inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells. FEMS Microbiol Lett 300:146–152

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by research grants from INRA and the Ministry of Education, Youth and Sport of the Czech Republic (Grant No. MSMT6046137305). We are grateful to Tom Beresford for providing the strain DPC 4571.

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Authors and Affiliations

  1. Department of Dairy, Fat and Cosmetic Science, Institute of Chemical Technology, Prague, Czech Republic

    Iva Jebava

  2. CIRM-BIA, UMR 1253—Science et Technologie du Lait et de l’OEuf, Institut National de la Recherche Agronomique, Rennes, France

    Victoria Chuat, Sylvie Lortal & Florence Valence

Authors
  1. Iva Jebava
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  2. Victoria Chuat
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  3. Sylvie Lortal
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  4. Florence Valence
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Correspondence to Iva Jebava.

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Jebava, I., Chuat, V., Lortal, S. et al. Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli. Curr Microbiol 68, 551–557 (2014). https://doi.org/10.1007/s00284-013-0512-5

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