Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles

Abstract

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering virus-like particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the Epoisses cheese virome. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems. IMPORTANCEWhether bacterial viruses (phages) are necessary or not to maintain food ecosystem function is not clear. They could play a negative role by killing cornerstone species that are necessary for fermentation. But they might also be positive players, by preventing the overgrowth of unwanted species (e.g. food spoilers). To assess phages contribution to food ecosystem functioning, it is essential to set up efficient procedures for extracting viral particles in solid .

High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community

“…The bacterial composition of these starters is often well characterized, but little is known about their phage-bacteria growth dynamics, with phages normally treated as problems, as viral infections can negatively affect or even eliminate the starter culture during production. Only a few studies characterize the bacteriophage community composition in cheese samples using viral metagenomes, mostly using whey or cheese rind samples 32,33 , and a recent metaanalysis using 184 cheese microbial metagenomes identified a high abundance of phageassociated sequences 34 . Here, we report the first study of phage-bacteria dynamics present in an artisanal cheese produced using the backslopping method, as well as its endogenous starter culture, from seven distinct artisanal Canastra Cheese producers in Minas Gerais Gerais state, in Brazil.…”

Bacteriophage ecology of fermented foods: anything new under the sun?

“…Only few studies used such direct approaches to characterize the phage communities of fermented foods. Dugat-Bony et al, used interferometric light microscopy to determine the phage concentration on the surface of three cheese varieties and found that it ranged from 1×10 9 to 4×10 10 particles per gram at least [35]*. They also observed different morphotypes on Epoisses cheese with TEM, giving first indications about the complexity of phage communities present on the cheese surface.…”

“…Recently, the first metavirome of the cheese surface was described [35] using Epoisses cheese as an example. The viral fraction was obtained according to an optimized protocol involving filtration, PEG concentration and chloroform treatment.…”

Dairy streptococcal cell wall and exopolysaccharide genome diversity

“…Phages infecting S. thermophilus are currently differentiated into five groups termed the cos (or Moineauvirus) and pac (or Brussowvirus) [8], 5093 [9], 987 [10] and P738 [11] groups. Limited studies on the biodiversity of S. thermophilus phages have been undertaken [12][13][14], though phageome studies of cheese and cheese-associated (by)products have recently garnered increasing attention [15,16]. To elucidate the phage-host interactome, it is essential to understand the biodiversity of both phages and their hosts that are applied in an industrial context, and of the phage-and host-encoded components that contribute to these interactions.…”

“…To elucidate the phage-host interactome, it is essential to understand the biodiversity of both phages and their hosts that are applied in an industrial context, and of the phage-and host-encoded components that contribute to these interactions. Recently, protocols have been optimized for the extraction and analysis of phageomes relating to cheese and cheese wheys [15,16]. Phageome analysis of fermented foods and their by-products provides useful insights into the prevalence, diversity and abundance of phages present in these foods, particularly where undefined starter culture mixtures are applied.…”