Viral metagenomic analysis of the cheese surface: a comparative study of rapid procedures for extracting virus-like particles

Abstract

12Running Head: Viral extraction procedure for cheese virome analysis 13 14 #Address correspondence to Eric Dugat-Bony, eric.dugat-bony@inra.fr 15 16 KEYWORDS 17 Cheese rind, viral metagenomic, VLPs extraction procedure 18 2 ABSTRACT 19The structure and functioning of microbial communities from fermented foods, including 20 cheese, have been extensively studied during the past decade. However, there is still a lack of 21 information about both the occurrence and the role of viruses in modulating the function of 22 this type of spatially structured and solid ecosystems. Viral metagenomics was recently 23 applied to a wide variety of environmental samples and standardized procedures for 24 recovering virus-like particles from different type of materials has emerged. In this study, we 25 adapted a procedure originally developed to extract viruses from fecal samples, in order to 26 enable efficient virome analysis of cheese surface. We tested and validated the positive 27 impact of both addition of a filtration step prior to virus concentration and substitution of 28 purification by density gradient ultracentrifugation by a simple chloroform treatment to 29 eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a 30 vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the 31 subsequent clusters assembled from the virome were analyzed to assess those belonging to 32 putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best 33 procedure was then chosen, and used to describe the Epoisses cheese virome. This study 34 provides the basis of future investigations regarding the ecological importance of viruses in 35 cheese microbial ecosystems.36 37 IMPORTANCE 38Whether bacterial viruses (phages) are necessary or not to maintain food ecosystem function 39 is not clear. They could play a negative role by killing cornerstone species that are necessary 40 for fermentation. But they might also be positive players, by preventing the overgrowth of 41 unwanted species (e.g. food spoilers). To assess phages contribution to food ecosystem 42 functioning, it is essential to set up efficient procedures for extracting viral particles in solid 43 3 food matrix, then selectively sequence their DNA without being contaminated by bacterial 44 DNA, and finally to find strategies to assemble their genome out of metagenomic sequences.45 This study, using cheese rind surface as a model, describes a comparative analysis of 46 procedures for selectively extracting viral DNA from cheese and to efficiently characterize 47 49The cheese surface hosts dense and diverse microbial communities composed of bacteria, 50 yeasts and filamentous fungi. Composition of these communities has been studied for decades 51 (see (1) and (2) for reviews). With the help of high throughput sequencing techniques, we 52 now have detailed pictures of the communities present in a large panel of cheese varieties, and 53 from all over the world (3-6). However, like many other microbia...

Meta-analysis of cheese microbiomes highlights contributions to multiple aspects of quality

“…A high abundance of phage-associated sequences were identified across the cheeses, accounting for a predicted 19.76% of the population, although their abundance did vary between samples. It is worth noting that the DNA extraction method used in this study was not tailored specifically for the isolation of phage, as was done elsewhere 26 , and so the level of phage detected here might be an underrepresentation of the actual virome. Predation by phage is a factor that shapes the formation of the cheese microbiota 27 , and infection of starters is the principal cause of fermentation failures during cheese production 28 .…”