Milk is a biological fluid with a dynamic composition of micronutrients and bioactive molecules that serves as a vital nutrient source for infants. Milk composition is affected by multiple factors, including genetics, geographical location, environmental conditions, lactation phase, and maternal nutrition, and plays a key role in dictating its microbiome. This study addresses a less-explored aspect, comparing the microbial communities in human breast milk with those in mature milk from species that are used for milk consumption. Since mature animal milk is used as a supplement for both the infant (formula) and the child/adolescent, our main aim was to identify shared microbial communities in colostrum and mature human milk. Using 16S rRNA metagenomic sequencing, we focused on characterizing the milk microbiota in the Northern Greek population by identifying shared microbial communities across samples and comparing the relative abundance of prevalent genera. We analyzed ten human milk samples (from five mothers), with five collected three days postpartum (colostrum) and five collected thirty to forty days postpartum (mature milk) from corresponding mothers. To perform an interspecies comparison of human milk microbiota, we analyzed five goat and five bovine milk samples from a local dairy industry, collected fifty to seventy days after birth. Alpha diversity analysis indicated moderate diversity and stability in bovine milk, high richness in goat milk, and constrained diversity in breast milk. Beta diversity analysis revealed significant distinctions among mammalian species, emphasizing both presence/absence and abundance-based clustering. Despite noticeable differences, shared microbial components underscore fundamental aspects across all mammalian species, highlighting the presence of a core microbiota predominantly comprising the Proteobacteria, Firmicutes, and Actinobacteriota phyla. At the genus level, Acinetobacter, Gemella, and Sphingobium exhibit significant higher abundance in human milk compared to bovine and goat milk, while Pseudomonas and Atopostipes are more prevalent in animal milk. Our comparative analysis revealed differences and commonalities in the microbial communities of various mammalian milks and unraveled the existence of a common fundamental milk core microbiome. We thus revealed both species-specific and conserved microbial communities in human, bovine, and goat milk. The existence of a common core microbiome with conserved differences between colostrum and mature human milk underscores fundamental similarities in the microbiota of milk across mammalian species, which could offer valuable implications for optimizing the nutritional quality and safety of dairy products as well as supplements for infant health.

This study investigates the dynamic changes in milk nutritional composition and microbial communities in Tibetan sheep and goats during the first 56 days of lactation. Milk samples were systematically collected at five time points (D0, D7, D14, D28, D56) post-delivery. In Tibetan sheep, milk fat, protein, and casein contents were highest on D0, gradually decreased, and stabilized after D14, while lactose and galactose levels showed the opposite trend. Goat milk exhibited similar initial peaks, with significant changes particularly between D0, D7, D14, and D56. 16S rRNA gene sequencing revealed increasing microbial diversity in both species over the lactation period. Principal coordinates analysis identified distinct microbial clusters corresponding to early (D0-D7), transitional (D14-D28), and mature (D56) stages. Core phyla, including Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, dominated the milk microbiota, with significant temporal shifts. Core microbes like Lactobacillus, Leuconostoc, and Streptococcus were common in both species, with species-specific taxa observed (e.g., Pediococcus in sheep, Shewanella in goats). Furthermore, we observed a highly shared core microbiota in sheep and goat milk, including Lactobacillus, Leuconostoc, and Streptococcus. Spearman correlation analysis highlighted significant relationships between specific microbial genera and milk nutrients. For instance, Lactobacillus positively correlated with total solids, non-fat milk solids, protein, and casein, while Mannheimia negatively correlated with protein content. This study underscores the complex interplay between milk composition and microbial dynamics in Tibetan sheep and goats, informing strategies for livestock management and nutritional enhancement. KEY POINTS: • The milk can be classified into three types based on the microbiota composition • The changes of milk microbiota are closely related to the variations in nutrition • Filter out microbiota with species specificity and age specificity in the milk.

Cow milk microbiota has received increased attention in recent years, not only because of its importance for human health but also because of its effect on the quality and technological properties of milk. Several studies, therefore, have investigated the effect of various production factors on the microbial composition of milk. However, most of the previous studies considered a limited number of animals from experimental or single farm, which could have biased the results. Therefore, this study aimed to understand the effect of different alpine production systems on the compositional and microbiological quality of milk, considering commercial herds with different feeding intensities and cattle breeds. The results obtained in this work indicated that the month/season of sampling (July for summer or February for winter) more than farm, breed and cow diet exerted significant effects on cow milk parameters and microbiota. In particular, significant differences were observed for urea content in milk between sampling seasons. Differences in milk fat were mainly related to breed specific effects. From a microbiological point of view, statistically significant differences were found in presumptive lactic acid bacteria counts. Based on a culture-independent method, milk obtained in February harbored the highest number of Firmicutes (e.g., Lactobacillus) and the lowest number of Actinobacteria (e.g., Corynebacterium). Moreover, bacterial richness and diversity were higher in July/summer during alpine pasture season indicating a significant effect of pasture feed on the growth of bacterial communities. The results of this study highlighted the effect of month/season mainly related to differences in feeding management (e.g., access to pasture during vegetation period, concentrates supplementation) on composition and microbiota in milk.

Season and location have previously been shown to be associated with differences in the microbiota of raw milk, especially in milk from pasture-based systems. Here, we further advance research in this area by examining differences in the raw milk microbiota from several locations across Ireland over 12 months, and by investigating microbiota associations with climatic variables and chemical composition. Shotgun metagenomic sequencing was used to investigate the microbiota of raw milk collected from nine locations (n = 241). Concurrent chemical analysis of the protein, fat, lactose, total solids, nonprotein nitrogen contents, and titratable acidity (TA) of the same raw milk were performed. Although the raw milk microbiota was highly diverse, a core microbiota was found, with Pseudomonas_E, Lactococcus, Acinetobacter, and Leuconostoc present in all samples. Microbiota diversity significantly differed by season and location, with differences in seasonality and geography corresponding to 11.8% and 10.5% of the variation in the microbiota. Functional and antibiotic resistance profiles also varied across season and location. The analysis of other metadata revealed additional interactions, such as an association between mean daily air and grass temperatures with the abundance of spoilage taxa like Pseudomonas species. Correlations were identified between pathogenic, mastitis-related species, fat content, and the number of sun hours, suggesting a seasonal effect. Ultimately, this study expands our understanding of the interconnected nature of the microbiota, environment/climate variables, and chemical composition of raw milk and provides evidence of a season- and location-specific microbiota.
OBJECTIVE: The microbiota of raw milk is influenced by many factors that encourage or prevent the introduction and growth of both beneficial and undesirable microorganisms. The seasonal and geographical impacts on the microbial communities of raw milk have been previously seen, but the relationships with environmental factors and the chemical composition has yet to be investigated. In this year-long study, we found that while raw milk is highly diverse, a core microbiota was detected for Irish raw milk, with strong evidence of seasonal and geographical influence. We also found associations between groups of microorganisms, environmental factors, and milk composition, which expand current knowledge on the relationships between microbial and chemical composition and the climate. These results provide evidence for the development of a tool to allow for the prediction of raw milk quality and safety.

The human milk (HM) microbiota, a highly diverse microbial ecosystem, is thought to contribute to the health benefits associated with breast-feeding, notably through its impact on infant gut microbiota. Our objective was to further explore the role of HM bacteria on gut homeostasis through a \"disassembly/reassembly\" strategy. HM strains covering the diversity of HM cultivable microbiota were first characterized individually and then assembled in synthetic bacterial communities (SynComs) using two human cellular models, peripheral blood mononuclear cells and a quadricellular model mimicking intestinal epithelium. Selected HM bacteria displayed a large range of immunomodulatory properties and had variable effects on epithelial barrier, allowing their classification in functional groups. This multispecies characterization of HM bacteria showed no clear association between taxonomy and HM bacteria impacts on epithelial immune and barrier functions, revealing the entirety and complexity of HM bacteria potential. More importantly, the assembly of HM strains into two SynComs of similar taxonomic composition but with strains exhibiting distinct individual properties, resulted in contrasting impacts on the epithelium. These impacts of SynComs partially diverged from the predicted ones based on individual bacteria. Overall, our results indicate that the functional properties of the HM bacterial community rather than the taxonomic composition itself could play a crucial role in intestinal homeostasis of infants.

The connection between maternal microbiota and infant health has been greatly garnered interest for therapeutic purposes. The early resident microbiota perpetually exhibits much more flexibility as compared to that of the adults, and therefore, constant need of understanding the infant as well as maternal microbiota and their implications however has increased. In this review, we focus mainly on the diversity of overall maternal microbiota including the gut, vaginal, colostrum microbiota and how inflammatory markers fluctuate throughout the normal pregnancy as well in pregnancy with complications. The maternal body undergoes a cascade of physiological changes including hormonal, immunological and metabolic events to support the fetal development. These changes at the time of pregnancy have been correlated with alteration in the composition and diversity of maternal microbiota. Along with alteration in microbiome, the levels of circulatory cytokines fluctuate by complex network of inflammation, in order to prevent the fetal allograft throughout the pregnancy. The dynamic relationship of gut microbiota with the host and its immune system allows one to have greater insights of their role in pregnancy and newborn\'s health. Emerging evidence suggests that the vertical transmission of bacterial community from mother to newborn may begin in-utero which contributes in developing the immune system and infant gut microbiota.

Sahiwal cattle is an indigenous cattle breed of Pakistan and mastitis is one of the major problems faced by Sahiwal cattle which hinders its production potential. The study was designed to investigate the milk microbiota of healthy and mastitic Sahiwal cattle as part of a multistep project to develop probiotics for the mitigation and control of mastitis. Milk samples of Sahiwal cattle (healthy clinical mastitis and subclinical mastitis) reared under similar husbandry and management practices were processed for 16S rRNA gene base metagenomics analysis.
Results revealed that Proteobacteria were dominant in the healthy group and subclinical mastitis group (56.48% and 48.77%, respectively) as compared to the clinical mastitis group (2.68%). In contrast, Firmicutes were abundant in the clinical mastitis group (64%) as compared to the healthy and subclinical mastitis groups (15.87% and 38.98%, respectively). Dominant species assigned in the healthy group were Ignavibacterium album, Novosphingobium capsulatum, Akkermansia muciniphila and Lactobacillus fermentum.The clinical mastitis group was dominated by Streptococcus dysgalactiae and Corynebacterium bovis, while subclinical mastitis group included Lactobacillus fermentum and uncultured acidobacteriales and Akkermansia muciniphila as dominant species. Alpha diversity indices showed higher microbial diversity in the healthy group compared to the clinical and sub-clinical mastitis groups.
It is concluded that the milk microbiota of healthy sahiwal cattle has higher diversity and dominant taxa in the different groups may be used as signature microbes for mastitis susceptibility. Akkermansia muciniphila is one of candidate specie that was identified and may be used for development of probiotics.

Bacteria of the Leuconostoc genus are Gram-positive bacteria that are commonly found in raw milk and persist in fermented dairy products and plant food. Studies have already explored the probiotic potential of L. mesenteroides, but not from a probiogenomic perspective, which aims to explore the molecular features responsible for their phenotypes. In the present work, probiogenomic approaches were applied in strains F-21 and F-22 of L. mesenteroides isolated from human milk to assess their biosafety at the molecular level and to correlate molecular features with their potential probiotic characteristics. The complete genome of strain F-22 is 1.99 Mb and presents one plasmid, while the draft genome of strain F-21 is 1.89 Mb and presents four plasmids. A high percentage of average nucleotide identity among other genomes of L. mesenteroides (≥ 96%) corroborated the previous taxonomic classification of these isolates. Genomic regions that influence the probiotic properties were identified and annotated. Both strains exhibited wide genome plasticity, cell adhesion ability, proteolytic activity, proinflammatory and immunomodulation capacity through interaction with TLR-NF-κB and TLR-MAPK pathway components, and no antimicrobial resistance, denoting their potential to be candidate probiotics. Further, the strains showed bacteriocin production potential and the presence of acid, thermal, osmotic, and bile salt resistance genes, indicating their ability to survive under gastrointestinal stress. Taken together, our results suggest that L. mesenteroides F-21 and F-22 are promising candidates for probiotics in the food and pharmaceutical industries.

Recently, the use of antimicrobials on dairy farms has been significantly limited from both the legislative and consumer points of view. This study aims to check the efficacy of selective dry cow therapy (SDCT) versus blanket dry cow therapy (BDCT) on bovine udder in healthy animals. SDTC is when an antibiotic is administered only to infected cows, compared with BDCT, where all cows receive an antimicrobial, regardless of their infection status. The milk samples were collected from enrolled Holstein Friesian cows 7 days before dry-off (T0) and 10 days after calving (T1) to assess somatic cell count (SCC), intramammary infections (IMIs), and milk microbiota variation. After pre-drying sampling, cows are randomly assigned to the following treatments: internal teat sealant alone (ITS; 24 cows), which is a treatment in a cow that does not receive antibiotics in SDTC, or in combination with intramammary antibiotic treatment (A+ITS; 22 cows). Non-statistically significant results are found between the two treatment groups at T1 for SCC, milk yield, and alpha diversity in milk microbiota. A statistically (p < 0.033) T1 IMI decrease is reported in the A+ITS group, and a significant beta diversity analysis is shown between the two timepoints (p = 0.009). This study confirms the possibility of selective drying without new IMI risk or increased SCC at calving, considering healthy cows without contagious infections and SCC values >200,000 cells/mL in the previous lactation.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.