E.Dugat-Bory,et al Food Microbiology 85 (2020)103278 Table S3)and more i did n observe qual atiredh by order of imp ences in the abun sonia.En cus.Lactob t to the reducing the stoc and Halomonas,were also detected in the Epoisse ed the DNA content of a membrane n an an ical densi 3.8 The cheese surt iggmtcrobi ta is tant evol n during th tion (T erent microbial groups.Micr ctions rt of nt Z 2018.p9 )Half of vesicle reads mappcd to phage ine with g microbial asse blage in this peculiar env nt.Our re the pro ted se su d ct of viruses on the dynamic of the chees 10% tha in the global virom various f the nt type of h DNA from Ep he nrst ch se as a n rial fo t an or to the use (MDA)(DNA of n the fin d our kno ble in S3).MDA is widely d in e the opportunity erented food. unity pr Conflict of interest ret DNA s highly dom ed by The authors declare no conflict of interest (only 3 with relative abundance sting Funding e dive nt and equ a p This v otic factors (e.g pH,salt,rip milk and the ew dom fer the ma tion,or the is thus ated hy y et The same may Acknowledgements was composed of many contig haring )for n6,P335 and C2-like groups.Inte ngly,the s (http://)for providing computational resources and very upport whey in New Zea Appendix A.Supplementary dat 1of89 82 nm,ar tible with actis phage. m a p References se and w ted in the rind of the raversus untreated samples, Table S3) and more importantly, we did not observe qualitative differences in the abundances of viral contigs as a function of chloroform treatment (Fig. 5 and Fig. S4). Furthermore, adding chloroform treatment to the extraction procedure resulted in a significant enrichment of the virome in actual viral sequences while drastically reducing the proportion of plasmids and sequences from microbial origin (unmapped reads). For one Epoisses, we sequenced the DNA content of a membrane vesicle fraction, separated from phage particles on an analytical density gradient. Only 0.5% of the total virome DNA yield (3.8 ng out of 548 ng) was recovered in this fraction (Table S3). Compared to total vesicles counts, we estimated that at most a few percent of the mem￾brane vesicles contained DNA. Indeed, the transport of nucleic acids only constitutes one of many potential roles for membrane vesicles (Gill et al., 2018). Half of vesicle reads mapped to phage contigs, in line with the proposal that membrane vesicles serve as decoys and trap phages, as recently reported in Vibrio cholerae (Reyes-Robles et al., 2018). Thus, compared to total viral DNA, only 0.35% got trapped into such “de￾coys”. Another 10% of vesicles reads corresponded to predicted plas￾mids, a property already reported for Escherichia coli membrane vesicles (Yaron et al., 2000). The third category of DNA content (also around 10%) in this vesicle fraction was composed of circular contigs that might be plasmidic. Interestingly, their relative proportion was constant in the global viromes across the various protocols, irrespective of the chloroform treatment, suggesting they might not belong to vesicles. The rapid procedure presented in this study enabled to obtain suf- ficient quantity of viral DNA from Epoisses cheese (equivalent of 1.5 g as starting material for each protocol) for direct library preparation prior to sequencing, avoiding the use of multiple displacement ampli- fication (MDA) (DNA quantities obtained for all samples presented in this study are available in Table S3). MDA is widely used in viral me￾tagenomic studies due to the insufficient DNA material extracted from the samples (Thurber et al., 2009). However, amplification bias has been documented for this technique (Pinard et al., 2006) which might provoke distortion in the viral community profiles and making prefer￾able direct DNA sequencing when possible. The Epoisses virome analyzed in this study was highly dominated by few contigs (only 3 with relative abundance > 1%), contrasting with the viromes produced from whey mixture which generally contain a wide diversity of abundant and equally distributed contigs (Muhammed et al., 2017). Cheese is a peculiar environment where the high nutrient availability and strong abiotic factors (e.g. pH, salt, ripening tempera￾ture) select from a quite large reservoir of microbial diversity present in milk and the dairy environment, only few dominant populations (Irlinger et al., 2015). The resulting microbial community in cheese rind after the maturation step is thus highly dominated by few taxa (Dugat￾Bony et al., 2016; Wolfe et al., 2014). The same rule may apply to viruses but additional experiments, such as time-series virome analysis during an entire cheese production cycle (from milk to mature cheese) would be necessary to confirm this hypothesis. The Epoisses virome was composed of many contigs sharing high sequence identity with known Lactococcus lactis phages including the widely known 936, P335 and C2-like groups. Interestingly, the second most abundant one (28% of reads), named 949_Epvir1, was very similar to the genome of L. lactis phage 949, a virulent phage isolated from cheese whey in New Zealand, which is phylogenetically distant from those groups of commonly isolated L. lactis phages (Samson and Moineau, 2010). We also detected a phage morphotype whose char￾acteristics, namely a head of 89 nm and a long noncontractile tail of 482 nm, are compatible with L. lactis phage 949. Lactococcus lactis is used as starter in the manufacture of Epoisses cheese. In a previous work describing the microbial diversity in twelve french cheese vari￾eties, it was detected as the dominant bacterial taxa in the core of Epoisses cheese and was also highly detected in the rind (Dugat-Bony et al., 2016). In the same study, other dominant bacterial genera of the Epoisses cheese rind included not-deliberately inoculated taxa such as, by order of importance, Psychrobacter, Marinomonas, Vibrio, Pseu￾doalteromonas, Glutamicibacter, Mesonia, Enterococcus, Lactobacillus and Halomonas. Interestingly, phages potentially infecting some of such non-starter bacterial taxa, including Glutamicibacter (Epvir4, most abundant contig in the Epoisses virome, 63% of the reads), Pseu￾doalteromonas (Epvir8, third most abundant contig, 1.1% of the reads), Vibrio, Leuconostoc and Halomonas, were also detected in the Epoisses cheese virome. The cheese surface microbiota is in constant evolution during the ripening process, and is characterized by the successive development of different microbial groups. Microbial interactions between different species have been observed in cheese (Irlinger and Mounier, 2009; Kastman et al., 2016; Mounier et al., 2008; Zhang et al., 2018), pro￾viding the first elements of comprehension regarding biotic forces sustaining microbial assemblage in this peculiar environment. Our re￾sults indicate that many microbial species living on the cheese surface are also subjected to viral predation, and shed light on the need of careful evaluation of the impact of viruses on the dynamic of the cheese microbial ecosystem. 5. Conclusions In this study, we adapted a viral extraction protocol originally de￾veloped for fecal samples to the cheese matrix and evaluated its use￾fulness for subsequent virome analysis. We demonstrated its efficiency by extracting viruses from three different type of cheese and produced the first cheese surface virome using Epoisses cheese as a model. Our results emphasize the positive impact of chloroform treatment and, to a lesser extent filtration, on the final virome quality in the cheese context. In the future, we anticipate virome analysis will expand our knowledge on cheese microbial ecosystems and possibly give the opportunity to better understand the rules of microbial assembly that occur in this fermented food. Conflict of interest The authors declare no conflict of interest. Funding This work was conducted in the frame of the Virome Access project supported by the French National Institute for Agricultural Research (INRA) and the Meta-omics and Microbial Ecosystems (MEM) pro￾gramme. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Acknowledgements We are grateful to Martine and Claude Boccara for their invaluable help with the interferometer device. We thank the INRA GeT-PlaGE platform (https://get.genotoul.fr/la-plateforme/get-plage) for sequen￾cing the Epoisses virome and the INRA MIGALE bioinformatics platform (http://migale.jouy.inra.fr) for providing computational resources and support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fm.2019.103278. References Aherfi, S., Colson, P., La Scola, B., Raoult, D., 2016. 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