Seasonal and geographical impact on the Irish raw milk microbiota correlates with chemical composition and climatic variables.

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. IMPORTANCE: 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.

An optimized culturomics strategy for isolation of human milk microbiota.

Viable microorganisms and a diverse microbial ecosystem found in human milk play a crucial role in promoting healthy immune system and shaping the microbial community in the infant's gut. Culturomics is a method to obtain a comprehensive repertoire of human milk microbiota. However, culturomics is an onerous procedure, and needs expertise, making it difficult to be widely implemented. Currently, there is no efficient and feasible culturomics method specifically designed for human milk microbiota yet. Therefore, the aim of this study was to develop a more efficient and feasible culturomics method specifically designed for human milk microbiota. We obtained fresh samples of human milk from healthy Chinese mothers and conducted a 27-day enrichment process using blood culture bottles. Bacterial isolates were harvested at different time intervals and cultured on four different types of media. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we identified a total of 6601 colonies and successfully obtained 865 strains, representing 4 phyla, 21 genera, and 54 species. By combining CBA and MRS media, we were able to cultivate over 94.4% of bacterial species with high diversity, including species-specific microorganisms. Prolonged pre-incubation in blood culture bottles significantly increased the number of bacterial species by about 33% and improved the isolation efficiency of beneficial bacteria with low abundance in human milk. After optimization, we reduced the pre-incubation time in blood culture bottles and selected optimal picking time-points (0, 3, and 6 days) at 37°C. By testing 6601 colonies using MALDI-TOF MS, we estimated that this new protocol could obtain more than 90% of bacterial species, reducing the workload by 57.0%. In conclusion, our new culturomics strategy, which involves the combination of CBA and MRS media, extended pre-incubation enrichment, and optimized picking time-points, is a feasible method for studying the human milk microbiota. This protocol significantly improves the efficiency of culturomics and allows for the establishment of a comprehensive repertoire of bacterial species and strains in human milk.

Two human milk-like synthetic bacterial communities displayed contrasted impacts on barrier and immune responses in an intestinal quadricellular model.

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.

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

Composition and interaction of maternal microbiota with immune mediators during pregnancy and their outcome: A narrative review.

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.

Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

Introduction: The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. Methods: We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Results and discussion: Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Probiogenomics of Leuconostoc Mesenteroides Strains F-21 and F-22 Isolated from Human Breast Milk Reveal Beneficial Properties.

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.

Associations between the Bacterial Composition of Farm Bulk Milk and the Microbiota in the Resulting Swedish Long-Ripened Cheese.

The maturation of a traditional Swedish long-ripened cheese has shown increasing variation in recent years and the ripening time is now generally longer than in the past. While the cheese is reliant on non-starter lactic acid bacteria for the development of its characteristic flavour, we hypothesised that the observed changes could be due to variations in the microbiota composition and number of bacteria in the raw milk used for production of the cheese. To evaluate associations between microbiota in the raw milk and the resulting cheese, three clusters of commercial farms were created to increase variation in the microbiota of dairy silo milk used for cheese production. Cheese production was performed in three periods over one year. Within each period, milk from the three farm clusters was collected separately and transported to the cheese production facility. Following pasteurisation, the milk was processed into the granular-eyed cheese and matured at a dedicated cheese-ripening facility. For each cheese batch, farm bulk and dairy silo milk samples, a starter culture, early process samples and cheese samples from different stages of maturation (7-20 months) were collected and their microbiota characterised using 16S rRNA amplicon sequencing. The microbiota in the farm bulk milk differed significantly between periods and clusters. Differences in microbiota in dairy silo milk were observed between periods, but not between farm clusters, while the cheese microbiota differed between periods and clusters. The top 13 amplicon sequence variants were dominant in early process samples and the resulting cheese, making up at least 93.3% of the relative abundance (RA). Lactococcus was the dominant genus in the early process samples and, together with Leuconostoc, also dominated in the cheese samples. Contradicting expectations, the RA of the aroma-producing genus Lactobacillus was low in cheese during ripening and there was an unexpected dominance of starter lactic acid bacteria even at the later stages of cheese ripening. To identify factors behind the recent variations in ripening time of this cheese, future studies should address the effects of process variables and the dairy environment.

Investigation of milk microbiota of healthy and mastitic Sahiwal cattle.

BACKGROUND: 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: 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. CONCLUSION: 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.

Effect of the Selective Dry Cow Therapy on Udder Health and Milk Microbiota.

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.

Microbiota and Resistome Analysis of Colostrum and Milk from Dairy Cows Treated with and without Dry Cow Therapies.

This study investigated the longitudinal impact of methods for the drying off of cows with and without dry cow therapy (DCT) on the microbiota and resistome profile in colostrum and milk samples from cows. Three groups of healthy dairy cows (n = 24) with different antibiotic treatments during DCT were studied. Colostrum and milk samples from Month 0 (M0), 2 (M2), 4 (M4) and 6 (M6) were analysed using whole-genome shotgun-sequencing. The microbial diversity from antibiotic-treated groups was different and higher than that of the non-antibiotic group. This difference was more evident in milk compared to colostrum, with increasing diversity seen only in antibiotic-treated groups. The microbiome of antibiotic-treated groups clustered separately from the non-antibiotic group at M2-, M4- and M6 milk samples, showing the effect of antibiotic treatment on between-group (beta) diversity. The non-antibiotic group did not show a high relative abundance of mastitis-causing pathogens during early lactation and was more associated with genera such as Psychrobacter, Serratia, Gordonibacter and Brevibacterium. A high relative abundance of antibiotic resistance genes (ARGs) was observed in the milk of antibiotic-treated groups with the Cephaguard group showing a significantly high abundance of genes conferring resistance to cephalosporin, aminoglycoside and penam classes. The data support the use of non-antibiotic alternatives for drying off in cows.

Maternal Diet May Modulate Breast Milk Microbiota-A Case Study in a Group of Colombian Women.

There is increasing evidence that the diet and nutritional status of women during pregnancy and lactation can modulate the microbiota of their milk and, therefore, the microbiota of the infant. An observational, descriptive, and cross-sectional study was carried out in a group of lactating women. Dietary intake during gestation and the first trimester of lactation was evaluated, and the microbiota was analyzed by 16S ribosomal RNA (rRNA) sequencing using the Illumina platform. Globally, Streptococcus spp. (32%), Staphylococcus spp. (17.3%), Corynebacterium spp. (5.1%) and Veillonella spp. (3.1%) were the predominant bacterial genera. The consumption of simple carbohydrates in gestation (rho = 0.55, p ≤ 0.01) and lactation (rho = 0.50, p ≤ 0.01) were positively correlated with Enterobacter spp. In lactation, a negative correlation was observed between the intake of simple carbohydrates and the genus Bifidobacterium spp. (rho = -0.51 p ≤ 0.01); furthermore, a positive correlation was identified between the intake of folic acid and Akkermansia spp. (rho = 0.47, p ≤ 0.01). Amplicon sequence variants (ASVs) associated with the delivery mode, employment relationship, the baby's gender, birth weight, the Body Mass Index (BMI) of the breastfeeding woman, and gestational weight gain were recovered as covariates in a linear mixed model. The results of this research showed that the maternal nutritional status and diet of women during gestation and lactation could modulate the microbiota of breast milk.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

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.

Absence of changes in the milk microbiota during Escherichia coli endotoxin induced experimental bovine mastitis.

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation.

Microbiota members from body sites of dairy cows are largely shared within individual hosts throughout lactation but sharing is limited in the herd.

BACKGROUND: Host-associated microbes are major determinants of the host phenotypes. In the present study, we used dairy cows with different scores of susceptibility to mastitis with the aim to explore the relationships between microbiota composition and different factors in various body sites throughout lactation as well as the intra- and inter-animal microbial sharing. RESULTS: Microbiotas from the mouth, nose, vagina and milk of 45 lactating dairy cows were characterized by metataxonomics at four time points during the first lactation, from 1-week pre-partum to 7 months post-partum. Each site harbored a specific community that changed with time, likely reflecting physiological changes in the transition period and changes in diet and housing. Importantly, we found a significant number of microbes shared among different anatomical sites within each animal. This was between nearby anatomic sites, with up to 32% of the total number of Amplicon Sequence Variants (ASVs) of the oral microbiota shared with the nasal microbiota but also between distant ones (e.g. milk with nasal and vaginal microbiotas). In contrast, the share of microbes between animals was limited (< 7% of ASVs shared by more than 50% of the herd for a given site and time point). The latter widely shared ASVs were mainly found in the oral and nasal microbiotas. These results thus indicate that despite a common environment and diet, each animal hosted a specific set of bacteria, supporting a tight interplay between each animal and its microbiota. The score of susceptibility to mastitis was slightly but significantly related to the microbiota associated to milk suggesting a link between host genetics and microbiota. CONCLUSIONS: This work highlights an important sharing of microbes between relevant microbiotas involved in health and production at the animal level, whereas the presence of common microbes was limited between animals of the herd. This suggests a host regulation of body-associated microbiotas that seems to be differently expressed depending on the body site, as suggested by changes in the milk microbiota that were associated to genotypes of susceptibility to mastitis.

Comparison of bacterial profiles in human milk from mothers of term and preterm infants.

BACKGROUND: Reducing the disposal of donated human milk (HM) is important for efficient management of human milk banks (HMBs). The presence of bacteria growth is the main factor that contributes to the disposal of donated HM. The bacterial profile in HM is suspected to differ between term and preterm mothers, with HM from preterm mothers containing more bacteria. Thus, elucidation of the causes of bacterial growth in preterm and term HM may help to reduce the disposal of donated preterm HM. This study compared the bacterial profiles of HM between mothers of term infants and mothers of preterm infants. METHODS: This pilot study was conducted in the first Japanese HMB, which was initiated in 2017. This study analyzed 214 human milk samples (term: 75, preterm: 139) donated by 47 registered donors (term: 31, preterm: 16) from January to November 2021. Bacterial culture results in term and preterm HM were retrospectively reviewed in May 2022. Differences in total bacterial count and bacterial species count per batch were analyzed using the Mann-Whitney U test. Bacterial loads were analyzed using the Chi-square test or Fisher's exact test. RESULTS: The disposal rate did not significantly differ between term and preterm groups (p = 0.77), but the total amount of disposal was greater in the preterm group (p < 0.01). Coagulase-negative Staphylococci, Staphylococcus aureus, and Pseudomonas fluorescens were frequently found in both types of HM. Serratia liquefaciens (p < 0.001) and two other bacteria were present in term HM; a total of five types of bacteria, including Enterococcus faecalis and Enterobacter aerogenes (p < 0.001) were present in preterm HM. The median (interquartile range) total bacterial counts were 3,930 (435-23,365) colony-forming units (CFU)/mL for term HM and 26,700 (4,050-334,650) CFU/mL for preterm HM (p < 0.001). CONCLUSIONS: This study revealed that HM from preterm mothers had a higher total bacterial count and different types of bacteria than HM from term mothers. Additionally, preterm infants can receive nosocomial-infection-causing bacteria in the NICU through their mother's milk. Enhanced hygiene instructions for preterm mothers may reduce the disposal of valuable preterm human milk, along with the risk of HM pathogen transmission to infants in NICUs.

Longitudinal Changes in Milk Microorganisms in the First Two Months of Lactation of Primiparous and Multiparous Cows.

The present experiment was carried out to analyze the longitudinal changes in milk microorganisms. For this purpose, milk samples were collected from 12 healthy cows (n = 96; six primiparous cows and six multiparous cows) at eight different time points. The characteristics and variations in microbial composition were analyzed by 16S rRNA gene high-throughput sequencing. In the primiparous group, higher and more stable alpha diversity was observed in transitional and mature milk compared with the colostrum, with no significant difference in alpha diversity at each time point in the multiparous group. Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota were the most dominant phyla, and Pseudomonas, UCG-005, Acinetobacter, Vibrio, Lactobacillus, Bacteroides, Serratia, Staphylococcus, and Glutamicibacter were the most dominant genera in both primiparous and multiparous cow milk. Some typically gut-associated microbes, such as Bacteroides, UCG-005, and Rikenellaceae_RC9_gut_group, etc., were enriched in the two groups. Biomarker taxa with the day in time (DIM) were identified by a random forest algorithm, with Staphylococcus showing the highest degree of interpretation, and the difference in milk microbiota between the two groups was mainly reflected in 0 d-15 d. Additionally, network analysis suggested that there were bacteria associated with the total protein content in milk. Collectively, our results disclosed the longitudinal changes in the milk microbiota of primiparous and multiparous cows, providing further evidence in dairy microbiology.

Microbial Diversity and Correlation between Breast Milk and the Infant Gut.

The gut microbiota is significant for infants to grow and develop in the early stages of life. The breast milk microbiota directly or indirectly influences colonizing and the development of early infant intestinal microbiota. Therefore, we wanted to study the microbial diversity and correlation between breast milk and the infant gut. By sequencing the 16S rRNA V3-V4 regions of microbiome in infant feces 1, 14, 20, 30, and 90 days after delivery as well as those in breast milk using Illumina NovaSeq, we studied the component of microbiome in both human milk and infant stools, analyzed the diversity of microbiota, and explored the relationship between them. We found that the richest bacteria in breast milk were Acinetobacter, Stenotrophomonas, Sphingopyxis, Pseudomonas, and Streptococcus, with a small amount of Lactobacillus, Bifidobacterium, and Klebsiella. The infant feces were abundant in Bifidobacterium, Escherichia-Shigella, Klebsiella, Streptococcus, Serratia, Bacteroides, and Lactobacillus, with a small number of Acinetobacter and Pseudomonas. Acinetobacter, Bifidobacterium, Klebsiella, and Lactobacillus appeared in the breast milk and infant feces, suggesting that they were transferred from the breast milk to the infant's gut.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Dietary Live Yeast Supplementation Influence on Cow's Milk, Teat and Bedding Microbiota in a Grass-Diet Dairy System.

The supplementation of animal feed with microbial additives remains questioning for the traditional or quality label raw milk cheeses with regard to microbial transfer to milk. We evaluated the effect of dietary administration of live yeast on performance and microbiota of raw milk, teat skin, and bedding material of dairy cows. Two balanced groups of cows (21 primiparous 114 ± 24 DIM, 18 multiparous 115 ± 33 DIM) received either a concentrate supplemented with Saccharomyces cerevisiae CNCM I-1077 (1 × 1010 CFU/d) during four months (LY group) or no live yeast (C group). The microbiota in individual milk samples, teat skins, and bedding material were analysed using culture dependent techniques and high-throughput amplicon sequencing. The live yeast supplementation showed a numerical increase on body weight over the experiment and there was a tendency for higher milk yield for LY group. A sequence with 100% identity to that of the live yeast was sporadically found in fungal amplicon datasets of teat skin and bedding material but never detected in milk samples. The bedding material and teat skin from LY group presented a higher abundance of Pichia kudriavzevii reaching 53% (p < 0.05) and 10% (p < 0.05) respectively. A significant proportion of bacterial and fungal ASVs shared between the teat skin and the milk of the corresponding individual was highlighted.