Hostname: page-component-78c5997874-g7gxr Total loading time: 0 Render date: 2024-11-02T19:54:15.616Z Has data issue: false hasContentIssue false
  • English
  • Français

Evaluation of different primers for PCR-DGGE analysis of cheese-associated enterococci

Published online by Cambridge University Press:  02 March 2009

Petra Mohar Lorbeg ,
Andreja Čanžek Majhenič  and
Irena Rogelj
Petra Mohar Lorbeg*
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
Andreja Čanžek Majhenič
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
Irena Rogelj
Affiliation:
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Chair of Dairy Science, Groblje 3, 1230Domžale, Slovenia
*
*For correspondence; e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Enterococci represent an important part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis published so far, six primer pairs amplifying different variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Enterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstrates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distinctive DGGE fingerprints, but with multiple bands patterns; therefore, these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used.

Keywords

16S rDNAidentificationPCR-DGGE
Type
Research Article
Information
Journal of Dairy Research , Volume 76 , Issue 3 , August 2009 , pp. 265 - 271
Copyright
Copyright © Proprietors of Journal of Dairy Research 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Boutte, C, Grubisic, S, Balthasart, P & Wilmotte, A 2006 Testing of primers for the study of cyanobacterial molecular diversity by DGGE. Journal of Microbiological Methods 65 542–550CrossRefGoogle Scholar
Cocolin, L, Diez, A, Urso, R, Rantsiou, K, Comi, G, Bergmaier, I & Beimfohr, C 2007 Optimization of conditions for profiling bacterial populations in food by culture-independent methods. International Journal of Food Microbiology 120 100–109CrossRefGoogle ScholarPubMed
Cocolin, L, Innocente, N, Biasutti, M & Comi, G 2004 The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process. International Journal of Food Microbiology 90 83–91CrossRefGoogle Scholar
Cocolin, L, Manzano, M, Cantoni, C & Comi, G 2001 Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region to Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages. Applied and Environmental Microbiology 67 5113–5121CrossRefGoogle ScholarPubMed
Coppola, S, Blaiotta, G, Ercolini, D & Moschetti, G 2001 Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese. Journal of Applied Microbiology 90 414–420CrossRefGoogle ScholarPubMed
Domig, KJ, Mayer, HK & Kneifel, W 2003 Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp.: 1. Media for isolation and enumeration. International Journal of Food Microbiology 88 147–164CrossRefGoogle ScholarPubMed
Ercolini, D 2004 PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods 56 297–314CrossRefGoogle ScholarPubMed
Ercolini, D, Hill, PJ & Dodd, CER 2003 Bacterial Community Structure and Location in Stilton Cheese. Applied and Environmental Microbiology 69 3540–3548CrossRefGoogle ScholarPubMed
Ercolini, D, Moschetti, G, Blaiotta, G & Coppola, S 2001a Behavior of Variable V3 Region from 16S rDNA of Lactic Acid Bacteria in Denaturing Gradient Gel Electrophoresis. Current Microbiology 42 199–202CrossRefGoogle ScholarPubMed
Ercolini, D, Moschetti, G, Blaiotta, G & Coppola, S 2001b The Potential of a Polyphasic PCR-DGGE Approach in Evaluating Microbial Diversity of Natural Whey Cultures for Water-Buffalo Mozzarella Cheese Production: Bias of Culture-Dependent and Culture-Independent Analyses. Systematic and Applied Microbiology 24 610–617CrossRefGoogle ScholarPubMed
Fasoli, S, Marzotto, M, Rizzotti, L, Rossi, F, Dellaglio, F & Torriani, S 2003 Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiology 82 59–70CrossRefGoogle ScholarPubMed
Florez, AB & Mayo, B 2006 PCR-DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese. International Dairy Journal 16 1205–1210CrossRefGoogle Scholar
Fontana, C, Sandro Cocconcelli, P & Vignolo, G 2005a Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages. International Journal of Food Microbiology 103 131–142CrossRefGoogle ScholarPubMed
Fontana, C, Vignolo, G & Cocconcelli, PS 2005b PCR-DGGE analysis for the identification of microbial populations from Argentinean dry fermented sausages. Journal of Microbiological Methods 63 254–263CrossRefGoogle ScholarPubMed
Franz, CMAP, Holzapfel, WH & Stiles, ME 1999 Enterococci at the crossroads of food safety? International Journal of Food Microbiology 47 1–24CrossRefGoogle ScholarPubMed
Franz, CMAP, Stiles, ME, Schleifer, KH & Holzapfel, WH 2003 Enterococci in foods – a conundrum for food safety. International Journal of Food Microbiology 88 105–122CrossRefGoogle Scholar
Giraffa, G 2003 Functionality of enterococci in dairy products. International Journal of Food Microbiology 88 215–222CrossRefGoogle ScholarPubMed
Gurtler, V 1999 The role of recombination and mutation in 16S-23S rDNA spacer rearrangements. Gene 238 241–252CrossRefGoogle ScholarPubMed
Klein, G 2003 Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. International Journal of Food Microbiology 88 123–131CrossRefGoogle ScholarPubMed
Muyzer, G 1999 DGGE/TGGE a method for identifying genes from natural ecosystems. Current Opinion in Microbiology 2 317–322CrossRefGoogle ScholarPubMed
Muyzer, G, de Waal, EC & Uitterlinden, AG 1993 Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59 695–700CrossRefGoogle Scholar
Ogier, J-C, Son, O, Gruss, A, Tailliez, P & Delacroix-Buchet, A 2002 Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis. Applied and Environmental Microbiology 68 3691–3701CrossRefGoogle ScholarPubMed
Ogier, JC, Lafarge, V, Girard, V, Rault, A, Maladen, V, Gruss, A, Leveau, JY & Delacroix-Buchet, A 2004 Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology 70 5628–5643CrossRefGoogle ScholarPubMed
Randazzo, CL, Torriani, S, Akkermans, ADL, de Vos, WM & Vaughan, EE 2002 Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis. Applied and Environmental Microbiology 68 1882–1892CrossRefGoogle ScholarPubMed
Randazzo, CL, Vaughan, EE & Caggia, C 2006 Artisanal and experimental Pecorino Siciliano cheese: Microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses. International Journal of Food Microbiology 109 1–8CrossRefGoogle ScholarPubMed
Temmerman, R, Huys, G & Swings, J 2004 Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends in Food Science & Technology 15 348–359CrossRefGoogle Scholar
Temmerman, R, Scheirlinck, I, Huys, G & Swings, J 2003 Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology 69 220–226CrossRefGoogle ScholarPubMed
von Wintzingerode, F, Gobel, UB & Stackebrandt, E 1997 Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiology Reviews 21 213–229CrossRefGoogle ScholarPubMed
Walter, J, Tannock, GW, Tilsala-Timisjarvi, A, Rodtong, S, Loach, DM, Munro, K & Alatossava, T 2000 Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers. Applied and Environmental Microbiology 66 297–303CrossRefGoogle ScholarPubMed
Wu, Y, Hayes, VM, Osinga, J, Mulder, IM, Looman, MW, Buys, CH & Hofstra, RM 1998 Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Nucleic Acids Research 26 5432–5440CrossRefGoogle ScholarPubMed
Zarazaga, M, Tenorio, C, Del Campo, R, Ruiz-Larrea, F & Torres, C 2002 Mutations in Ribosomal Protein L16 and in 23S rRNA in Enterococcus Strains for Which Evernimicin MICs Differ. Antimicrobial Agents and Chemotherapy 46 3657–3659CrossRefGoogle ScholarPubMed