Mother\'s milk contains a unique microbiome that plays a relevant role in offspring health. We hypothesize that maternal malnutrition during lactation might impact the microbial composition of milk and affect adequate offspring gut colonization, increasing the risk for later onset diseases. Then, Wistar rats were fed ad libitum (Control, C) food restriction (Undernourished, U) during gestation and lactation. After birth, offspring feces and milk stomach content were collected at lactating day (L)4, L14 and L18. The V3-V4 region of the bacterial 16S rRNA gene was sequenced to characterize bacterial communities. An analysis of beta diversity revealed significant disparities in microbial composition between groups of diet at L4 and L18 in both milk, and fecal samples. In total, 24 phyla were identified in milk and 18 were identified in feces, with Firmicutes, Proteobacteria, Actinobacteroidota and Bacteroidota collectively representing 96.1% and 97.4% of those identified, respectively. A higher abundance of Pasteurellaceae and Porphyromonas at L4, and of Gemella and Enterococcus at L18 were registered in milk samples from the U group. Lactobacillus was also significantly more abundant in fecal samples of the U group at L4. These microbial changes compromised the number and variety of milk-feces or feces-feces bacterial correlations. Moreover, increased offspring gut permeability and an altered expression of goblet cell markers TFF3 and KLF3 were observed in U pups. Our results suggest that altered microbial communication between mother and offspring through breastfeeding may explain, in part, the detrimental consequences of maternal malnutrition on offspring programming.

Milk is one of the most nutritionally complete foods and plays an important role in the human diet. Buffalo milk represents 15% of worldwide milk production and is an important source of bioactive compounds. Buffalo milk has a great market in the Mediterranean area, and dairy products, such as Mozzarella and Ricotta di Bufala Campana, obtained with the Italian Mediterranean buffalo milk, are acknowledged with the Protected Designation of Origin (PDO). This study aimed to characterize, using high-throughput sequencing of the 16S rRNA gene, the milk core microbiome of water buffalo rises in the Amaseno Valley included in the Mozzarella PDO region. The principal features of the core and the auxiliary buffalo milk microbiome are the predominance of Firmicutes and Lactococcus, one of the most important lactic acid bacteria (LAB) taxa in the dairy industry. The comparative analysis of the core microbiomes indicated that the milk of the Italian Mediterranean Buffalo and other mammals share the presence of Streptococcus-affiliated OTUs (operational taxonomic units). Our data also demonstrated that the core microbiome of milk samples collected from PDO and non-PDO regions differ in the number and type of taxa. KEY POINTS: • Buffalo milk and their derivate products are becoming more popular worldwide. • Dairy locations and practice management affect the structure of the milk microbiota. • Next-generation sequencing (NGS) analysis allows to identify the features of the Italian Buffalo milk microbiome.

Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony-forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α- and β-diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

The milk microbiota and mediated metabolites directly affect the health of the udder in dairy cows. Inulin, a dietary prebiotic, can modulate the profile of gastrointestinal microbiota. However, whether the inulin intake affects the milk microbial population and metabolites remains unknown. In this study, 40 subclinical mastitis (SCM) cows were randomly divided into 5 groups. Five inulin addition doses, 0, 100, 200, 300, and 400 g/day per cow, based on the same basal diet, were supplemented. The experiments lasted for 8 weeks. The results showed lower relative abundance of mastitis-causing and proinflammation microbes in milk (i.e., Escherichia-Shigella, Pseudomonas, Rhodococcus, Burkholderia-Caballeronia-Paraburkholderia, etc.) and higher abundances of probiotics and commensal bacteria, such as Lactobacillus, Bifidobacterium, etc., in the cows fed 300 g/day inulin compared to that in the control group. Meanwhile, the levels of arachidonic acid proinflammatory mediators (leukotriene E3, 20-carboxy-leukotriene B4, and 12-Oxo-c-LTB3) and phospholipid metabolites were reduced, and the levels of compounds with antibacterial and anti-inflammatory potential (prostaglandin A1, 8-iso-15-keto-prostaglandin E2 [PGE2], etc.) and participating energy metabolism (citric acid, l-carnitine, etc.) were elevated. These data suggested that inulin intake might modulate the microflora and metabolite level in extraintestinal tissue, such as mammary gland, which provided an alternative for the regulation and mitigation of SCM. IMPORTANCE The profile of the microbial community and metabolic activity in milk are the main determinants of udder health status and milk quality. Recent studies have demonstrated that diet could directly modulate the mammary gland microbiome. Inulin is a probiotic dietary fiber which can improve the microbiota population in the gastrointestinal tract. However, whether inulin intake can further regulate the profile of the microbiota and metabolic activities in milk remains unclear. In subclinical mastitic cows, we found that inulin supplementation could reduce the abundance of Escherichia-Shigella, Pseudomonas, Rhodococcus, and Burkholderia-Caballeronia-Paraburkholderia and the levels of (±)12, 13-DiHOME, leukotriene E3 and 20-carboxy-leukotriene B4 etc., while it elevated the abundance of Lactobacillus, Bifidobacterium, and Muribaculaceae, as well as the levels of prostaglandin A1 (PGA1), 8-iso-15-keto-PGE2, benzoic acid, etc. in milk. These data suggest that inulin intake affects the profile of microorganisms and metabolites in milk, which provides an alternative for the regulation of mastitis.

Professionals in animal agriculture promote prudent use of antimicrobials to address public and animal health concerns, such as reduction of antimicrobial residues and antimicrobial resistance (AMR) in products. Few studies evaluate the effect of selective dry-cow therapy on preservation of the milk microbiome or the profile of AMR genes (the resistome) present at freshening. Our objectives were to characterize and compare the microbiomes and resistomes in the colostrum of cows with low somatic cell count that were treated or not treated with intramammary cephapirin benzathine at dry-off. From a larger parent study, cows on a New York dairy farm eligible for dry-off and with histories of somatic cell counts ≤200,000 cells/mL were enrolled to this study (n = 307). Cows were randomly assigned to receive an intramammary antimicrobial and external teat sealant (ABXTS) or sealant only (TS) at dry-off. Composite colostrum samples taken within 4 h of freshening, and quarter milk samples taken at 1 to 7 d in milk were subjected to aerobic culture. The DNA extraction was performed on colostrum from cows with culture-negative samples (ABXTS = 43; TS = 33). The DNA from cows of the same treatment group and parity were pooled (26 pools; ABXTS = 12; TS = 14) for 16S rRNA metagenomic sequencing. Separately, the resistome was captured using a custom RNA bait library for target-enriched sequencing. Sequencing reads were aligned to taxonomic and AMR databases to characterize the microbiome and resistome, respectively. The R statistical program was used to tabulate abundances and to analyze differences in diversity measures and in composition between treatment groups. In the microbiome, the most abundant phyla were Firmicutes (68%), Proteobacteria (23%), Actinobacteria (4%), and Bacteroidetes (3%). Shannon and richness diversity means were 0.93 and 14.7 for ABXTS and 0.94 and 13.1 for TS, respectively. Using analysis of similarities (ANOSIM), overall microbiome composition was found to be similar between treatment groups at the phylum (ANOSIM R = 0.005), class (ANOSIM R = 0.04), and order (ANOSIM R = -0.04) levels. In the resistome, we identified AMR gene accessions associated with 14 unique mechanisms of resistance across 9 different drug classes in 14 samples (TS = 9, ABXTS = 5). The majority of reads aligned to gene accessions that confer resistance to aminoglycoside (TS = ABXTS each 35% abundance), tetracycline (TS = 22%, ABXTS = 54%), and β-lactam classes (TS = 15%, ABXTS = 12%). Shannon diversity means for AMR class and mechanism, respectively, were 0.66 and 0.69 for TS and 0.19 and 0.19 for ABXTS. Resistome richness diversity means for class and mechanism were 3.1 and 3.4 for TS and 1.4 and 1.4 for ABXTS. Finally, resistome composition was similar between groups at the class (ANOSIM R = -0.20) and mechanism levels (ANOSIM R = 0.01). Although no critical differences were found between treatment groups regarding their microbiome or resistome composition in this study, a larger sample size, deeper sequencing, and additional methodology is needed to identify more subtle differences, such as between lower-abundance features.

The bovine udder is colonized by a huge quantity of microorganisms that constitute the intramammary ecosystem, with a specific role in modulating not only udder homeostasis and mastitis susceptibility, but also the quality of the dairy products. However, generating high-quality bacterial DNA can be critical, especially starting from a complex biological matrix like milk, characterized by high fat, protein, and calcium contents. Here, bacterial DNA was recovered from a commercial ultra-high-temperature (UHT) milk sample artificially spiked with a predetermined mock community composition and from three bulk tank milk (raw milk) samples. The DNA was isolated using three different protocols to evaluate the effect of the extraction procedures on the milk microbiota composition. In the mock community experiment, the bacterial profiles generated by the three DNA extraction protocols were profoundly different, with the genera Staphylococcus, Lactobacillus, Listeria, and Salmonella underestimated by all the protocols. Only one protocol revealed values close to the expected abundances for Escherichia/Shigella spp., Bacillus spp., Enterococcus spp., and Pseudomonas spp. On the other hand, the nonspiked UHT milk sample exhibited a similar microbiota composition, revealing the prevalence of Acinetobacter spp., for all the DNA extraction protocols. For the raw milk samples, the three DNA extraction kits performed differently, revealing significant separations in both the microbial richness (alpha diversity) and composition (beta diversity). Our study highlights the presence of significant differences among these procedures, probably due to the different DNA extracting capacities and to the different properties of the milk samples, revealing that the selection of DNA extraction protocol is a critical point. IMPORTANCE The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals. This study provides evidence that milk, as other complex sources, could be critical for generating high-quality DNA for microbiota analysis. In addition, it demonstrates that the microbial population highlighted by metagenomic studies changes in relation to different DNA extraction procedures, revealing that attention should be paid especially when comparing different studies.

Bovine mastitis is a widespread infectious disease. In addition to the economic damages associated with reduced milk yield due to mastitis, the problem of food contamination by microorganism metabolites, in particular toxins, is also a concern. Horizontal transfer of microorganisms from animal populations to humans can also be complicated by antibiotic resistance. Therefore, bovine mastitis is relevant to the study of microbiology and veterinary medicine. In this study, we investigated the microbiome of milk samples from healthy cows and cows with different forms of mastitis from individual quarters of the udder of cows during first and second lactation. Total DNA was extracted from milk samples. The V3-V4 regions of the bacterial 16S rRNA genes from each sample were amplified to generate a library via high-throughput sequencing. We revealed significant dominance of several operational taxonomic units (OTUs) corresponding mostly to groups of Staphylococcus aureus, Aerococcus spp., and Streptococcus spp. In addition, we unexpectedly identified Streptococcus thermophilus in samples with high SCC quantities. We found some infectious agents that characterized summer mastitis. We demonstrated that in Central Russia, mastitis is associated with a wide variety of causal organisms. We observed some differences in the diversity of the two investigated farms. However, we did not find any significant difference among healthy, mastitis and subclinical samples according to their SCC status from either farms by principal component analysis. Linear discriminant analysis effect size (LEfSe) confirmed the presence of several indicator genera in farms from Moscow and the Tula Region. These results confirm the complex bacterial etiology of bovine mastitis.

Analysis of milk micrbiomes from healthy cows and cows with different (clinical and subclinical) forms of mastitis was performed at two farms of the Central Russia. An increase in the operational taxonomic units (OTUs) of bacteria of the phylum Proteоbacteria belonging primarily to Pseudomonadales, Burkholderiales, as well as Streptococcaceae, Staphylococcaceae, and Bacillaceae in the animals with mastitis was detected. The Planococcaceae OTU percentage decreased. The ratio of rarely presented OTUs also changed in the milk of animals with mastitis.

Breast milk is an unbeatable food that covers all the nutritional requirements of an infant in its different stages of growth up to six months after birth. In addition, breastfeeding benefits both maternal and child health. Increasing knowledge has been acquired regarding the composition of breast milk. Epidemiological studies and epigenetics allow us to understand the possible lifelong effects of breastfeeding. In this review we have compiled some of the components with clear functional activity that are present in human milk and the processes through which they promote infant development and maturation as well as modulate immunity. Milk fat globule membrane, proteins, oligosaccharides, growth factors, milk exosomes, or microorganisms are functional components to use in infant formulas, any other food products, nutritional supplements, nutraceuticals, or even for the development of new clinical therapies. The clinical evaluation of these compounds and their commercial exploitation are limited by the difficulty of isolating and producing them on an adequate scale. In this work we focus on the compounds produced using milk components from other species such as bovine, transgenic cattle capable of expressing components of human breast milk or microbial culture engineering.