title: Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening.
abstract: DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem. Gouda cheese manufacturing is a proper model to evaluate the use of PMA for selective detection of intact cells since large fraction of membrane compromised cells emerges as a background in the cheese matrix during ripening. In this study, the effect of PMA on cheese community profiles was evaluated throughout manufacturing and ripening using quantitative PCR (qPCR). PMA effectively inhibited the amplification of DNA derived from membrane compromised cells and enhanced the analysis of the intact fraction residing in the cheese samples. Furthermore, a two-step protocol, which involves whole genome amplification (WGA) to enrich the DNA not modified with PMA and subsequent sequencing, was developed for the selective metagenome sequencing of viable fraction in the Gouda cheese microbial community. The metagenome profile of PMA treated cheese sample reflected the viable community profile at that time point in the cheese manufacturing.
authors.affiliation: NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands; Wageningen University, Laboratory of Microbiology, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
authors.forename: Oylum
authors.initials: O
authors.lastname: Erkus
authors.affiliation: Wageningen University, Laboratory of Microbiology, Dreijenplein 10, 6703 HB Wageningen, The Netherlands; Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Victor C L
authors.initials: VC
authors.lastname: de Jager
authors.affiliation: Wageningen University, Laboratory of Food Microbiology, Bornse Weilanden 9, PO Box 17, 6700 AA Wageningen, The Netherlands.
authors.forename: Renske T C M
authors.initials: RT
authors.lastname: Geene
authors.affiliation: NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands.
authors.forename: Ingrid
authors.initials: I
authors.lastname: van Alen-Boerrigter
authors.affiliation: NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands.
authors.forename: Lucie
authors.initials: L
authors.lastname: Hazelwood
authors.affiliation: NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands; Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Sacha A F T
authors.initials: SA
authors.lastname: van Hijum
authors.affiliation: NIZO food research BV, P.O. Box 20, 6710 BA Ede, The Netherlands; Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands; Wageningen University, Host Microbe Interactomics Group, De Elst 1, 6708 WD Wageningen, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
authors.affiliation: Top Institute Food and Nutrition (TIFN), Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 557, 6700 AN, Wageningen, The Netherlands; Wageningen University, Laboratory of Food Microbiology, Bornse Weilanden 9, PO Box 17, 6700 AA Wageningen, The Netherlands. Electronic address: eddy.smid@wur.nl.
authors.forename: Eddy J
authors.initials: EJ
authors.lastname: Smid
mesh_terms: D001386:Azides / Q000494:pharmacology*; D002611:Cheese / Q000382:microbiology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology / Q000379:methods*; D050296:Microbial Viability; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001386:Azides; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: Cheese; Dairy; Food fermentation; Metagenomics; Microbial community profiling; Propidium monoazide