The objectives of this study were to explore bacterial community assembly from cow teat skin to raw milk cheeses and to evaluate the role of farming systems on this assembly using 16S rRNA gene high-throughput sequencing. The two grazing systems studied (extensive vs. semi-extensive) had a greater effect on the microbiota of cow teat skin than on that of raw milks and cheeses. On teat skin, the relative abundance of several taxa at different taxonomic levels (Coriobacteriia, Bifidobacteriales, Corynebacteriales, Lachnospiraceae, Atopobium, and Clostridium) varied depending on the grazing system and the period (early or late summer). In cheese, the abundance of sub-dominant lactic acid bacteria (LAB) varied depending on the grazing system. Overall, 85% of OTUs detected in raw milks and 27% of OTUs detected in ripened cheeses were also found on cow teat skin. Several shared OTUs were assigned to taxa known to be involved in the development of cheese sensory characteristics, such as Micrococcales, Staphylococcaceae, and LAB. Our results highlight the key role of cow teat skin as a reservoir of microbial diversity for raw milk, and for the first time, that cow teat skin serves as a potential source of microorganisms found in raw-milk cheeses.

An exhaustive analysis was performed on more than 2000 microbiotas from French Protected Designation of Origin (PDO) cheeses, covering most cheese families produced throughout the world. Thanks to a complete and accurate set of associated metadata, we have carried out a deep analysis of the ecological drivers of microbial communities in milk and “terroir” cheeses. We show that bacterial and fungal microbiota from milk differed significantly across dairy species while sharing a core microbiome consisting of four microbial species. By contrast, no microbial species were detected in all ripened cheese samples. Our network analysis suggested that the cheese microbiota was organized into independent network modules. These network modules comprised mainly species with an overall relative abundance lower than 1%, showing that the most abundant species were not those with the most interactions. Species assemblages differed depending on human drivers, dairy species, and geographical area, thus demonstrating the contribution of regional know-how to shaping the cheese microbiota. Finally, an extensive analysis at the milk-to-cheese batch level showed that a high proportion of cheese taxa were derived from milk under the influence of the dairy species and protected designation of origin.

In dairy cattle, teat disinfection at the end of milking is commonly applied to limit colonization of the milk by pathogenic microorganisms via the teat canal. The post-milking products used can irritate the teat skin and unbalance its microbial population. Our study aimed to assess the impact of different milking products on the balance of the microbial communities on the teat skin of cows and in their milk. For 12 weeks at the end of each milking operation, three groups of seven Holstein dairy cows on pasture received either a chlorhexidine gluconate-based product (G) or a hydrocolloidal water-in-oil emulsion (A), or no post-milking product (C). The composition of the bacterial and fungal communities on the teat skin and in the milk were characterized using a culture-dependent method and by high-throughput sequencing of marker genes to obtain amplicon sequence variants (ASVs). The individual microbiota on the cows’ teat skin was compared for the first time to that of a cow pool. In contrast to the milk, the post-milking treatment influenced the microbiota of the teat skin, which revealed a high microbial diversity. The water-in-oil emulsion appeared to slightly favour lactic acid bacteria and yeasts and to limit the development of undesirable bacteria such as Pseudomonas and Staphylococcus.

A previous study identified differences in rind aspects between Cantal-type cheeses manufactured from the same skimmed milk, supplemented with cream derived either from pasture-raised cows (P) or from cows fed with maize silage (M). Using an integrated analysis of multiomic data, the present study aimed at investigating potential correlations between cream origin and metagenomic, lipidomic and volatolomic profiles of these Cantal cheeses. Fungal and bacterial communities of cheese cores and rinds were characterized using DNA metabarcoding at different ripening times. Lipidome and volatolome were obtained from the previous study at the end of ripening. Rind microbial communities, especially fungal communities, were influenced by cream origin. Among bacteria, Brachybacterium were more abundant in P-derived cheeses than in M-derived cheeses after 90 and 150 days of ripening. Sporendonema casei, a yeast added as a ripening starter during Cantal manufacture, which contributes to rind typical aspect, had a lower relative abundance in P-derived cheeses after 150 days of ripening. Relative abundance of this fungus was highly negatively correlated with concentrations of C18 polyunsaturated fatty acids and to concentrations of particular volatile organic compounds, including 1-pentanol and 3-methyl-2-pentanol. Overall, these results evidenced original interactions between milk fat composition and the development of fungal communities in cheeses.

Adding massive amounts of lactic starters to raw milk to manage the sanitary risk in the cheese-making process could be detrimental to microbial diversity. Adjusting the amount of the lactic starter used could be a key to manage these adverse impacts. In uncooked pressed cheeses, we investigated the impacts of varying the doses of a lactic starter (the recommended one, 1×, a 0.1× lower and a 2× higher) on acidification, growth of Staphylococcus aureus SA15 and Shiga-toxin-producing Escherichia coli (STEC) O26:H11 F43368, as well as on the bacterial community patterns. We observed a delayed acidification and an increase in the levels of pathogens with the 0.1× dose. This dose was associated with increased richness and evenness of cheese bacterial community and higher relative abundance of potential opportunistic bacteria or desirable species involved in cheese production. No effect of the increased lactic starter dose was observed. Given that sanitary criteria were paramount to our study, the increase in the pathogen levels observed at the 0.1× dose justified proscribing such a reduction in the tested cheese-making process. Despite this, the effects of adjusting the lactic starter dose on the balance of microbial populations of potential interest for cheese production deserve an in-depth evaluation.

The bio-preservation potential of Lactococcus garvieae lies in its capacity to inhibit the growth of staphylococci, especially S. aureus, in dairy products and in vitro. In vitro, inhibition is modulated by the level of aeration, owing to hydrogen peroxide (H2O2) production by L. garvieae under aeration. The S. aureus response to this inhibition has already been studied. However, the molecular mechanisms of L. garvieae underlying the antagonism against S. aureus have never been explored. This study provides evidence of the presence of another extracellular inhibition effector in vitro. This effector was neither a protein, nor a lipid, nor a polysaccharide, nor related to an L-threonine deficiency. To better understand the H2O2-related inhibition mechanism at the transcriptome level and to identify other mechanisms potentially involved, we used RNA sequencing to determine the transcriptome response of L. garvieae to different aeration levels and to the presence or absence of S. aureus. The L. garvieae transcriptome differed radically between different aeration levels mainly in biological processes related to fundamental functions and nutritional adaptation. The transcriptomic response of L. garvieae to aeration level differed according to the presence or absence of S. aureus. The higher concentration of H2O2 with high aeration was not associated with a higher expression of L. garvieae H2O2-synthesis genes (pox, sodA, spxA1) but rather with a repression of L. garvieae H2O2-degradation genes (trxB1, ahpC, ahpF, gpx). We showed that L. garvieae displayed an original, previously undiscovered, H2O2 production regulation mechanism among bacteria. In addition to the key factor H2O2, the involvement of another extracellular effector in the antagonism against S. aureus was shown. Future studies should explore the relation between H2O2-metabolism, H2O2-producing LAB and the pathogen they inhibit. The nature of the other extracellular effector should also be determined.