Human Milk Microbiome—A Review of Scientific Reports

One of the most important bioactive components of breast milk are free breast milk oligosaccharides, which are a source of energy for commensal intestinal microorganisms, stimulating the growth of Bifidobacterium, Lactobacillus, and Bacteroides in a child’s digestive tract. There is some evidence that maternal, perinatal, and environmental-cultural factors influence the modulation of the breast milk microbiome. This review summarizes research that has examined the composition of the breast milk microbiome and the factors that may influence it. The manuscript highlights the potential importance of the breast milk microbiome for the future development and health of children. The origin of bacteria in breast milk is thought to include the mother’s digestive tract (entero-mammary tract), bacterial exposure to the breast during breastfeeding, and the retrograde flow of breast milk from the infant’s mouth to the woman’s milk ducts. Unfortunately, despite increasingly more precise methods for assessing microorganisms in human milk, the topic of the human milk microbiome is still quite limited and requires scientific research that takes into account various conditions.


Introduction
In recent years, the World Health Organization (WHO) has recommended exclusive breastfeeding until the child is 6 months old and continuing with complementary feeding until the age of 2 or longer if, due to the health-promoting properties of human milk, the mother and child need it [1].
Natural feeding ensures the child's optimal health and promotes proper development.Infants fed with human milk are less likely to suffer from infectious diseases, necrotizing enterocolitis, and sudden infant death syndrome (SIDS), die less often, and are additionally at a lower risk of developing the following in the future: overweight, obesity, type I and II diabetes, asthma, lymphoma, Hodgkin's disease, lymphocytic leukemia, and myeloid hypercholesterolemia [1][2][3][4].Breastfeeding also protects the mother.The immediate benefits of breastfeeding include a shorter period of bleeding after childbirth, faster uterine involution, lactation infertility, faster weight loss, and a lower level of depression [1,5].However, it also has long-term beneficial effects, including a lower risk of breast cancer, ovarian cancer, osteoporosis, as well as a lower incidence of hip fractures, hypertension, diabetes, and hyperlipidemia [1,6].
Human milk is the mother's most valuable gift to her child due to the health-promoting properties resulting from its composition.It is a colloidal solution of carbohydrates, fats, proteins, vitamins, growth factors, microelements, and other biologically active chemical compounds, the composition of which changes depending on the phases of lactation, the health of women, their physiology, race, or the geographical environment in which they live.Diet minimally affects the content of human milk components [7][8][9].One of the most important bioactive components of human milk are free human milk oligosaccharides (HMOs), a type of prebiotic.HMOs are an energy source for commensal intestinal microbes.They stimulate the growth of Bifidobacterium, Lactobacillus, and Bacteroides in the infant's digestive tract and are also believed to prevent neonatal diarrhea and respiratory infections [10,11].The concentration of HMOs ranges from 20 to 25 g/L in colostrum and from 5 to 20 g/L in mature milk, which makes them the third-largest solid component in human milk [10,12].
The nutritional properties of human milk have been known for hundreds of years.Currently, human milk is treated as a medicine and has many clinical applications.This is related to its numerous health properties, i.e., anti-inflammatory, immunomodulatory, antioxidant, anti-infective, as well as its impact on the development of the digestive system [13][14][15][16][17][18][19][20][21].
In addition to a number of substances and nutrients, human milk contains a wide range of microorganisms [22] (Figure 1).The dominant bacteria in breast milk belong to the genera Streptococcus, Staphylococcus, Micrococcus, Lactococcus, Lactobacillus, and Bifodobacterium [17].Consumption of breast milk has a positive effect on the colonization of the child's intestinal microbiota, with a predominance of the genera Bifidobacterium and Lactobacillus.In turn, in children fed with formula milk, it was shown that there are fewer bifidobacteria and lactobacilli in the microbiota, despite the fact that the child had contact with these bacteria during vaginal delivery.Please note that microorganisms present in milk may play an important role in the development of the gastrointestinal microbiome of infants [23,24].The transfer of many lactic acid bacteria from a breastfeeding mother is a natural mechanism that improves the development of the infant's intestinal microbiota, which is associated with increased immunity [25].The composition of the breast milk microbiome has a huge impact on the health of the baby, which is presented later in the manuscript.Therefore, considering the benefits of breastfeeding and the possible difficulties that breastfeeding women may experience, every effort should be made to support women during lactation.The role of midwives is crucial at this time because, as specialists in breastfeeding promotion, they have a great influence on mothers' decisions about how to feed their children.Midwives are responsible not only for providing specialist help but also for increasing the sense of competence in their abilities when breastfeeding.[25,26]).(HMOs-human milk oligosaccharides; HAMLET-human a-lactalbumin made lethal to tumor cells; BMI-body mass index; NICU-neonatal intensive care unit; NECnecrotizing enterocolitis).
Recent research by Boix-Amorós et al. [27], using molecular techniques, estimated that an infant may consume approximately 10 7 to 10 8 bacterial cells per day through mother's milk (~800 mL).However, can we define the basic composition of human milk?Previous research has shown the presence of viruses, bacteria, and fungi in human milk [22].The dominant types of bacteria have been distinguished, but the composition of milk is variable and depends on many factors (Figures 1 and 2).So far, it has been shown that the "core" microbiome of human milk consists of the following genera: Streptococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacterium, Ralstonia, Cutibacterium, Sphingomonas, and Bradyrhizobium, and their abundance varies depending on the sample  [25,26]).(HMOs-human milk oligosaccharides; HAMLET-human a-lactalbumin made lethal to tumor cells; BMI-body mass index; NICU-neonatal intensive care unit; NEC-necrotizing enterocolitis).
Recent research by Boix-Amorós et al. [27], using molecular techniques, estimated that an infant may consume approximately 10 7 to 10 8 bacterial cells per day through mother's milk (~800 mL).However, can we define the basic composition of human milk?Previous research has shown the presence of viruses, bacteria, and fungi in human milk [22].The dominant types of bacteria have been distinguished, but the composition of milk is variable and depends on many factors (Figures 1 and 2).So far, it has been shown that the "core" microbiome of human milk consists of the following genera: Streptococcus, Staphylococcus, Serratia, Pseudomonas, Corynebacterium, Ralstonia, Cutibacterium, Sphingomonas, and Bradyrhizobium, and their abundance varies depending on the sample (Figure 2) [28][29][30][31].Metagenomic analysis of human milk by sequencing performed by Ward et al. [32] showed that human milk contains over 360 prokaryotic genera, including Proteobacteria (65%) and Firmicutes (34%) as the dominant types and Pseudomonas spp.(61.1%),Staphylococcus spp.
(33.4%), and Streptococcus spp.(0.5%) as the dominant bacterial species.It should be emphasized that a normal microbiome also includes sequences related to fungi, protozoa, and viruses [33].According to McGuire MK and McGuire MA [34], human milk is probably mother nature's prototypical probiotic food-providing a cocktail of microorganisms, the amount of which depends on various factors and is necessary for the newborn during the critical period of growth and development.
Nutrients 2024, 16, x FOR PEER REVIEW 4 of 39 (Figure 2) [28][29][30][31].Metagenomic analysis of human milk by sequencing performed by Ward et al. [32] showed that human milk contains over 360 prokaryotic genera, including Proteobacteria (65%) and Firmicutes (34%) as the dominant types and Pseudomonas spp.(61.1%),Staphylococcus spp.(33.4%), and Streptococcus spp.(0.5%) as the dominant bacterial species.It should be emphasized that a normal microbiome also includes sequences related to fungi, protozoa, and viruses [33].According to McGuire MK and McGuire MA [34], human milk is probably mother nature's prototypical probiotic foodproviding a cocktail of microorganisms, the amount of which depends on various factors and is necessary for the newborn during the critical period of growth and development.

Origin of Microbiota in Human Milk
Colonization of human milk with microorganisms is a dynamic, complex, and not fully understood process.Until recently, it was believed that every bacterial cell present in human milk was the result of contamination of the mother's skin or the baby's oral cavity.Only the detection of live bacterial cells or DNA of anaerobic species in human milk, which cannot survive in aerobic conditions and are usually found in the intestinal environment, initiated a discussion among researchers on the hypothetical sources of the origin of bacteria in human milk [35], as shown in Figure 3. Research is still on going to determine whether the mammary gland is home to the so-called "mucosal surface model" or the "continuous flow model."

Origin of Microbiota in Human Milk
Colonization of human milk with microorganisms is a dynamic, complex, and not fully understood process.Until recently, it was believed that every bacterial cell present in human milk was the result of contamination of the mother's skin or the baby's oral cavity.Only the detection of live bacterial cells or DNA of anaerobic species in human milk, which cannot survive in aerobic conditions and are usually found in the intestinal environment, initiated a discussion among researchers on the hypothetical sources of the origin of bacteria in human milk [35], as shown in Figure 3. Research is still on going to determine whether the mammary gland is home to the so-called "mucosal surface model" or the "continuous flow model".

Child's Mouth Cavity
By analyzing the retrograde flow of human milk from the baby's mouth into the milk ducts, it is likely that microorganisms from the baby's mouth can seed the human milk bacterial microbiome.This hypothesis is supported by the presence of bacteria in human Figure 3. Overview of hypothetical sources of the human milk microbiome.Data is from references [22,27,[35][36][37][38].

Child's Mouth Cavity
By analyzing the retrograde flow of human milk from the baby's mouth into the milk ducts, it is likely that microorganisms from the baby's mouth can seed the human milk bacterial microbiome.This hypothesis is supported by the presence of bacteria in human milk that are characteristic of the oral environment: Streptococcus salivarius, Streptococcus mitis, Rothia mucilaginosa, and Gemella spp.[22].The importance of direct breastfeeding and the reverse flow of human milk from the baby's mouth to the milk ducts was presented in the study of Biaga et al.The study included prematurely born children (gestational age 32-34 weeks) who initially received human milk from a bottle.Only after the child's general condition and proper sucking function were stabilized did the children begin to be fed directly from the breast.It was observed that the human milk microbiome after the transition from feeding with expressed human milk using a bottle to direct breastfeeding became more diverse and dominated by typical oral microorganisms, i.e., Streptococcus and Rothia spp., thus confirming that contact with the baby's oral cavity shapes the human milk microbiome [36].However, research by Ruiz et al. [39] presents the opposite situation, in which oral bacteria found in human milk colonize the child's oral cavity.The authors of this study found that there is a very large share of taxa in common between colostrum obtained from pregnant women before delivery and samples collected from the oral cavity of a newborn.Interestingly, in 8 of 10 pairs (mother-child), both isolates were > 99.9% identical at the nucleotide level.The results of these studies suggest that at least some of the bacteria typical of the oral cavity (the largest percentage of Streptococcus spp.and Staphylococcus spp.) enter the child's oral cavity during breastfeeding.These data strongly suggest that some bacteria colonize the infant's oral microbiome from human milk [39].It is very likely that human milk bacteria and baby's oral bacteria exchange characteristics, thus creating both environments during breastfeeding.According to the literature, the oral microbiome of breastfed children differs significantly from the oral microbiome of children fed with formula.These studies highlight that the choice of how to feed a child influences the development of the oral microbiome, which may ultimately impact both short-and long-term health effects [40].

Mother's Skin
Human milk contains many bacteria of the Staphylococcus genus, including commensal bacteria typical of human skin: S. epidermidis, S. hominis, S. haemolyticus, and S. lugdunensis.Jiménez et al. [33], conducting research on 23 women and their children (16 breastfed and 7 fed with formula), showed that S. epidermidis dominated both in human milk and in the feces of breastfed infants.In turn, in infants fed with formula milk, S. epidermidis was less prevalent, which was a feature that differentiated both study groups [41].Human skin commensal bacteria such as Cutibacterium acnes and species of the Corynebacterium genus are also often identified in human milk.Moreover, the human skin commensal Malassezia spp. is the main genus of fungi present not only in human milk, but also in and around the sebaceous glands [42].Therefore, it can be assumed that the bacterial microbiota of the skin colonize the mammary gland by passing through the nipple.Pannaraj et al., in their research on a group of 107 healthy mother-child pairs, proved that 27.7% of gastrointestinal bacteria came from human milk, and another 10.3% from the skin of the areola [43].
It is certainly necessary to continue research in this area to assess the contribution of the mother's skin bacterial microbiota to the microbiome of the child's gastrointestinal tract and to the microbiome of human milk.

Maternal Digestive Tract
The literature on the subject indicates that the microbiome of human milk has common features with the microbiome of the gastrointestinal tract of a breastfeeding mother.The genus Saccharomyces, which includes some of the most abundant fungi identified in the gastrointestinal tract, is also one of the main types of fungi present in human milk [44].Human milk also contains the following types of bacteria: Bifidobacterium, Veillonella, Bacteroides, Parabacteroides, Clostridium, Collinsella, Faecalibacterium, Coprococcus, and Blautia [33,[45][46][47][48][49][50].These bacteria are anaerobes that would not survive in the aerobic conditions of the child's mouth and on the mother's skin.Hence, the hypothetical "entero-mammary route", which would explain the presence of the previously mentioned bacteria in human milk.Dendritic cells take up live bacteria by disrupting the intestinal epithelium.Then, for several days in the mesenteric lymph nodes, these cells sequester live bacteria, which are transferred through the lymphatic system to individual parts of the body, including the mammary gland [22].Perez et al. [51], in studies on a group of mice, showed that bacteria could be identified 60% more often in the mesenteric lymph nodes of pregnant mice compared to mice that were not pregnant.The obtained results suggest that the ability of bacteria to translocate increases during lactation [51].During this period, the number of lymphatic vessels in the mammary gland tissues increases.This is confirmed by research conducted on a group of mice that were orally administered two genetically modified strains of lactic acid bacteria (Lactococcus lactis MG1614 and Lactobacillus salivarius PS2).These bacteria were detected in milk and mammary tissue in a group of pregnant mice but not in a control group [52].Subsequent studies indicate that some species of bacteria typical of the gastrointestinal tract (Escherichia coli, Faecalibacterium, and Eubacterium spp.) occasionally enter the mammary gland.Hence, the conclusion that only some bacteria have the ability to get from the digestive tract to the mammary gland [51].It is also important that the microbiome of human milk can be modified, e.g., by diet or pre/probiotics used by a breastfeeding woman.Jiménez et al. [33] used a daily oral dose of Lactobacillus salivarius CECT5713 and Lactobacillus gasseri CECT5714 or a placebo in a group of breastfeeding women with diagnosed mastitis.At the beginning of the study, the average number of staphylococci in human milk in both groups was comparable (probiotic group 4.74 and control group 4.81 log 10 colony forming unit (CFU)/mL).However, on the 30th day of the study, the average number of Staphylococcus spp. in the probiotic group (2.96 log 10 CFU/mL) was significantly lower than in the control group (4.79 log 10 CFU/mL), and L. salivarius CECT5713 and L. gasseri CECT5714 could be isolated from human milk samples in 6 out of 10 cases.Moreover, on day 14 of the study, no symptoms of mastitis were observed in the group of women using probiotics, and in women from the control group, symptoms of mastitis persisted throughout the study period [53].In turn, when it comes to the impact of a breastfeeding woman's diet on the microbiome of human milk, it has been proven that the gastrointestinal microbiota of breastfed children depends on the mother's vegetarian diet [54].

Microorganisms in Human Milk
The microbiome of human milk is associated with a large bacterial diversity; the normal microbiome also includes sequences associated with viruses, fungi, and protozoa [33].To date, data on the composition of human milk are related to the use of classical microbiological methods.However, the development of molecular biology techniques has made it possible to demonstrate the presence of non-culturable microorganisms from milk, which has significantly expanded the knowledge of the composition of human milk [32,33].

Archaea
The use of the latest molecular techniques has allowed Archaea to be demonstrated in human milk.Previous research has also revealed the DNA of archaea and methanogenic microorganisms in human milk, including Haloarcula marismortui, Halorhabdus utahensis, and Halomicrobium mukohataei [33].In turn, Togo et al. [55] showed the presence of Methanobrevibacter smithii in both colostrum (3 samples) and milk (5 samples).In contrast, Methanobrevibacter oralis was cultured from a single sample of human milk [55].Data about Archaea have been largely underestimated in human milk microbiome assessments due to the technical difficulty of the methods used in and their evaluation [56].

Viruses
Data on the human milk virome are limited [57].Research so far has focused mainly on DNA viruses, leaving aside RNA viruses due to diagnostic difficulties [22].
Available research results indicate the presence of bacteriophages in human milk.Viruses accumulate in the gastrointestinal tract during the first two years of life, indicating environmental acquisition [22].However, the concentration of bacteriophages in the digestive tract of infants is highest in the first days of life and decreases with age [58].The analysis conducted by Pannaraj et al. showed that bacteriophages dominated in both infant feces (95.5% ± 3.2%) and mother's milk (95.2 ± 2.8%).In the same study, a small percentage (4.5 ± 3.2% and 4.8 ± 2.8% of infants and mothers, respectively) in the feces were eukaryotic viruses.The dominant family in infant feces was Siphoviridae, while in mother's milk samples-viruses from the Myoviridae family [59].Duranti et al. showed common bifidophages in infant and maternal fecal samples and human milk.Researchers concluded that bifidophages are transmitted to infants during breastfeeding [57].In turn, non-phage viral sequences present in human milk samples were identified as members of the families Papillomaviridae, Retroviridae, and Herpesviridae (including the genus Cytomegalovirus) [33].Pannaraj et al. found that a high percentage of bacteriophages that are transmitted from the mother's milk to the infant's digestive tract may contribute to shaping the microbiome of the infant's digestive tract [43].
Human milk may be a carrier of vertical transmission of some viruses, including human immunodeficiency virus (HIV), cytomegalovirus, and human T-cell leukemia [60][61][62].An intriguing result was the demonstration of the presence of Ebolavirus RNA in the milk of mothers who did not report any symptoms of the disease.It has been suggested that mother's milk was the source of the Ebola virus to the infant who died from the disease [63].Transmission of SARS-CoV-2 through human milk has not been demonstrated so far [64,65].

Bacteria
The microbiome of human milk has a diversity of bacterial genera and species.With the development of sequencing methods, the DNA of many non-culturable species has been demonstrated [32,33].Defining the core composition of the microbiome is extremely difficult, as many factors influence its modulation [34].The dominant bacteria in breast milk belong to the genera Streptococcus, Staphylococcus, Micrococcus, Lactococcus, Lactobacillus, and Bifodobacterium, but the presence of different species in human milk is variable.[17].
It is also important to estimate the number of bacteria that are present in human milk.Boix-Amorós et al. [27] estimated that an infant may consume approximately 10 7 to 10 8 bacterial cells per day through mother's milk (~800 mL).A study conducted by Jiménez et al. showed that the number of bacteria in human milk (based on 10 samples) ranged from 2.24 to 2.62 log 10 CFU/mL.However, the ratio of human DNA to microbial DNA was approximately 9:1 [33].These data show that microbiota make up a significant percentage of human milk, indicating their important role.
Microbiota diversity also depends on the stage of lactation.It was shown that the following genera predominate in colostrum samples: Weisella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus.However, in samples collected during the 6-month feeding period, the dominant species were Veillonella, Leptotrichia, and Prevotella [48] (Figure 2).The higher species diversity in colostrum may be due to the higher concentration of colostrum nutrients.Soto et al. showed that S. epidermidis was present in 77.27% of the samples, and the Streptococcus genus (dominant species: S. mitis, S. salivarius, and S. parasanguinis) was present in 60.61% of the tested samples [71].Recently, a new species isolated from human milk was Streptococcus lactarius [69].In turn, Jiménez et al. showed that Staphylococcus aureus was the dominant species in milk samples from women with acute mastitis.However, in women suffering from subacute mastitis, the predominance of S. epidermidis has been demonstrated [33].In turn, the presence of Enterococcus spp. in human milk is variable and ranges from 0.5 to 4.0% of the total bacterial DNA [46,48].
Probiotic bacteria have also been identified in human milk.The presence of probiotic strains in human milk has a positive effect on the development of the intestinal microbiota of newborns.It should be emphasized that some strains of lactic acid bacteria (LAB) isolated from human milk have shown the ability to inhibit the growth of pathogenic bacteria.The mechanism of action is based mainly on the production of antimicrobial bacteriocins by LAB, among other things [72,73].It should be emphasized that Lactobacillus bacteria present in milk may inhibit the growth of strains of the following genera: Pseudomonas, Escherichia, and Serratia [74].The strains with probiotic properties isolated so far included L. gasseri, L. salivarius, L. fermentum (inducing the production of interleukin 10) [75], and L. rhamnosus (exhibiting antioxidant and anticancer properties) [76].Colostrum samples were found to have a higher percentage of Bifidobacterium or Lactobacillus (76.9% and 48.6%, respectively) compared to mature milk samples [77].
The microbiome of human milk is variable and shaped by many factors.The qualitative and quantitative composition of bacteria in milk is constantly changing [37] and is influenced by many factors.For this reason, this section of the paper provides an overview of the basic human milk microbiome.Characterization of the composition of the microbiome, depending on various factors, is presented later in the manuscript.

Fungi
The presence of fungi in human milk is estimated at approximately 10 5 CFU/mL [26].The dominant genera in milk include Saccharomyces, Malassezia, Alternaria, Rhodotorula, and Candida [26,31].The presence of fungi of the Basidiomycota and Ascomycota genera in most milk samples was demonstrated by Jiménez et al. [33].The tested samples showed the presence of the following genera and species: Caloceracornea, Guepiniopsisbuccina, Malasseziaglobosa, Podosporaanserina, Sordariamacrospora, Candida dubliniensis, Malasseziarestricta, Talaromycesstipitatus, and Yarrowialipolytica [33].In turn, Boix-Amorós et al. [44] showed that 89% of tested milk samples from healthy women had detectable levels of fungal DNA, with an estimated median load of 3.5 × 10 5 CFU/mL.Among the samples examined (pyrosequencing), they also showed that the dominant genus was Malassezia (44%), followed by Candida (19%) and Saccharomyces (12%).Additionally, researchers have assessed the impact of ingredients present in milk on the presence of fungi.A positive correlation was found between Malassezia spp.and the bacterial load, as well as between this genus and lactose.However, a positive correlation was demonstrated for Candida spp.and milk protein [44].The origin of fungi in human milk is unknown.It should be emphasized that the species isolated in the study by Boix-Amorós et al. [44] are present on human skin, but also in other niches on the body (including Malassezia, Candida, Aspergillus, and Penicillium), or isolated from the human intestine (including in.Candida, Malassezia, Cladosporium, or Debaromyces) [81][82][83].Due to its lipophilic nature, the genus Malassezia colonizes the seborrheic parts of the skin and is maintained by the use of fatty acids present in sebum [84].These properties may promote the survival and growth of Malassezia spp. in milk, which is characterized by high levels of fat.Boix-Amorós et al. [44] showed that the genera identified among four female populations were Malassezia, Davidiella, Sistotrema, and Penicillium.In contrast, the genera Wallemia and Aspergillus were found only in samples of women from Finland [42].Further work should indicate the origin of fungi present in milk and their impact on the health of newborns and the development of the microbiome.

Protozoa
There is limited data regarding protozoa in human milk.The first data on the possibility of parasite transmission (Toxoplasma gondii) with breast milk were presented by Bonametti et al. [85].Guého et al. [84] concluded that breastfeeding could be a source of T. gondii in the case of a mother who was ill with toxoplasmosis.Jiménez et al. [33] showed the presence of T. gondii in 35% of the tested samples and Giardia intestinalis in only one milk sample (10%).In contrast, Khamsian et al. [86] showed that out of 300 human milk samples, 1 sample (0.3%) was positive for T. gondii.
Chagas disease (infection with the protozoan Trypanosoma cruzi) is a major parasitic disease in the Americas and one of the main neglected tropical diseases [87].To date, only two studies, from 1936 [88] and 1983 [89], have shown the presence of trypomastigotes in the milk of mothers who were in the acute phase of Chagas disease.However, in 1993, a colleague of Prof. Mazza published a letter to the editor [88], in which he corrected the issue of transmission of T. cruzi with breast milk.Jörg [90] corrected that the milk collected by Mazza et al. [88] was contaminated with blood, indicating that transmission of T. cruzi was via blood (bleeding nipples).Similar results related to the transmission of T. cruzi via nipple digestion were presented by Mediana-Lopez [91].Data on the transmission of T. cruzi through breastfeeding in humans are scarce.The data are not up-to-date and are carried out on a small number of patients.Nevertheless, there are suspicions about the possible transmission of T. cruzi with breast milk, so this topic should be further analyzed.
Much of the data relate to the presence of antibodies in breast milk that protect the infant from infection, among others: Giardia lamblia [92], Strongyloides stercoralis [93], Plasmodium falciparum [94], or Onchocerca volvulus [95].

Factors Influencing the Microbiome of Human Milk
According to the literature, genetic factors, nutrition of a breastfeeding woman, method of delivery, stage of lactation, time of day, and geographical factors influence the composition of human milk.It is believed that the above-mentioned factors can modulate the microbiota, including the mother's skin or intestines, as well as the infant's microbiota.Therefore, maternal, perinatal, environmental, and cultural factors may influence the milk microbiome.Currently, there is no detailed analysis of the human milk microbiome and no detailed information on the impact of factors on the human food microbiome.Knowledge of a given topic and possible modulation of the human milk microbiota could have an impact on the colonization of microorganisms in infants, as well as on the development of the infants' immune system.

The Influence of Environmental and Cultural Factors on the Microbiome of Human Milk
Geographical location is also among the factors that may influence the composition of the microbiome.Both the area of residence and the presence of urban and rural areas influence the qualitative and quantitative composition of human milk.
A higher number of bacteria was found in the milk of women from rural areas.Taghizadeh et al. showed that the median number of Lactobacillus bacteria was higher in women living in rural areas than in women living in urban areas [96].Also, Sinkiewicz et al. showed that the milk of women living in rural areas is characterized by a higher number of lactobacilli (1.3 × 10 3 CFU/mL) compared to women living in cities (3.0 × 10 2 CFU/mL).Moreover, in women living in rural areas, they showed the presence of L. reuteri in 14.0% of the samples, while in women from urban areas, the same species was found in 15.0% of the samples [97].
Geographical location also influences the composition of the microbiome.For example, higher numbers of lactobacilli have been found in women in Israel, South Africa, Japan, and South Korea [97].In turn, González et al. showed that the most frequently isolated genera from mother's milk among Mozambican women were Staphylococcus (96.4%),Streptococcus (92.7%), and Lactobacillus (56.4%).However, HIV RNA was detected in 24.0% of the milk samples.Researchers have also observed higher bacterial diversity and the prevalence of Lactobacillus spp. in milk samples from HIV-positive women [98].In turn, a study conducted by Urbaniak et al. using sequencing (Ion Torrent) confirmed that the milk of healthy women living in Canada was dominated by the genera Actinobacter, Stenotrophomonas, Pseudomonas, Streptococcus, and Staphylococcus.However, the milk of Caucasian women living in Canada was dominated by Pseudomonas, Streptococcus, and Lactobacillus [99].Dave et al. showed that Streptococcus was the dominant genus in the milk of Mexican-American women [100].However, Boix-Amorós et al. showed that the milk microbiota among Spanish women was dominated by the following genera: Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter.Importantly, researchers have shown that the median bacteria in human milk is approximately 10 6 CFU/mL, suggesting that breastfed infants consume approximately 7-8 million bacterial cells per day [27].A study by Gonzalez et al. involving lactating women from Guatemala showed that ten different species of the Staphylococcus genus were associated with early lactation, including S. hominis, S. epidermidis, and S. hyicus.The dominant species of the Streptococcus genus identified were S. mitis, S. parasanguinis, S. peroris, S. pneumoniae, S. pseudopneumoniae, and S. salivarius.Other species identified in milk during early lactation included Corynebacterium tuberculostearicum, C. jeikeium, L. gasseri, Acinetobacter johnsonii, Kocuria palustris, and Janthinobacterium agaricidamnosum.In turn, bacteria associated with the late phase of lactation among breast feeding women from Guatemala included species of the genera Staphylococcus and Streptococcus (which were associated with the early phase of lactation) and species such as Sphingobium yanoikuyae, Pseudomonas putida, Stenotrophomonas maltophilia, Ottowia beijingensis, and Comamonas testosteroni [101].
The analysis conducted by Kumar et al. showed the highest level of Bacteroidetes in Spanish women (natural childbirth) compared to women from other countries (Finland, South Africa, and China).However, women from South Africa showed a significantly higher abundance of Proteobacteria compared to other samples.The samples from Finnish women had higher levels of Firmicutes and lower levels of Proteobacteria when compared to the samples from other countries (p = 0.004).The highest levels of Actinobacteria were observed among mothers from China who gave birth vaginally.At the genus level, Chinese women had higher levels of Streptococcus, and Spanish women had higher levels of Cutibacterium and Pseudomonas.Lactobacillaceae was found exceptionally in samples from Finland, Bifidobacteriace was found only in South African women, and Enterococcaceae was found in samples from all countries except China [102].However, Davé et al. showed that the predominant genera in milk from Mexican-American women were Streptococcus, Staphylococcus, Xanthomonadaceae, and Sediminibacterium [100].
Lackey et al. showed that Staphylococcus and Streptococcus bacteria were present among all analyzed milk samples (regardless of the women's place of residence).However, the genus Rhizobium was detected in the milk of women in a rural community in Ethiopia.In turn, among the samples of milk from women from Peru, it was shown that 50% of the bacterial community was the Streptococcus genus, and this level was significantly higher compared to the other examined women's communities (from Africa and the United States).Interestingly, milk from women from the Washington site had higher levels of Dyella spp.[103].
Data on microbiome diversity depending on geographical location are presented in Table 1.A study by Boix-Amorós et al. [42] showed that geographical location did not affect the amount of fungi in human milk, but there were differences in qualitative assessment.Researchers confirmed that samples from South African women had significantly higher levels of Ascomycota spp.and lower levels of Basidiomycota spp.and Malassezia spp.than in other population studies.In turn, milk samples from Chinese women showed lower levels of fungi from the genus Penicillium and Rhodotorula, while the genus Saccharomyces was more abundant in milk samples from Spanish and Finnish women compared to other geographical locations [42].• women who exclusively breastfed had a higher percentage of Streptococcus parasanguis compared to women who also used formula; • women who were HIV positive had a higher absolute number of Lactobacillus spp.Cacho et al. [115] South America (Brazil) 47 women (of which two with mastitis); urban

MALDI-TOF MS
• the total number of bacteria in breast milk ranged from 1.5 to 4.0 log 10 CFU/mL; • high number of bacteria in colostrum; • among LAB the following were isolated: L. gasseri, Bifidobacterium breve, and S. salivarius.

The Influence of Maternal Factors on the Microbiome of Human Milk
Maternal factors affecting the human milk microbiome include age, body mass index (BMI), taking medications, type of diet, supplementation, physical activity, lifestyle, stimulants, and diseases accompanying breastfeeding women (Table 2) [7,[117][118][119].
The stage of lactation has a significant impact on the formation of milk microbiota.Cabrera-Rubio et al. [48] compared the microbiota of colostrum and mature milk collected 1 and 6 months after delivery.Bacteria from the genera Firmicutes, Weissella, and Leuconostoc (Lactobacillales) predominated in colostrum, followed by Staphylococcus, Streptococcus, and Lactococcus.These genera were also abundant in samples collected at a later stage, but the numbers of the genera Veillonella (phylum Firmicutes), Leptotrichia (Fusobacteria), and Prevotella (Bacteroidetes) increased [48].However, there are studies that do not confirm this relationship.Li et al. showed that the milk microbiome at 3.6 months and above 6 months of lactation does not show diversity [7].
The mother's health shapes the microbiota of human milk, in particular the woman's obesity and diseases accompanying breastfeeding women (e.g., celiac disease, allergy).Maternal obesity reduces microbial diversity, the number of Bifidobacterium spp.and Lactobacillus and increases the number of Staphylococcus [120].Other results were presented by Moosavi et al., indicating that BMI and maternal cigarette smoking do not affect the human milk microbiome [45].Similar results were obtained by Li et al. [121].
There are limited data on the impact of infection and comorbidities of breastfeeding women on the microbiota of human milk.Currently, the impact of celiac disease, HIV, ormastitis on the composition of human milk, including the microbiome, has been described [98,107].Mastitis is a state of dysbiosis in which the number of bacteria increases to one million CFU/mL.Research by scientists from Spain showed that milk samples from 1849 women suffering from acute, subacute, or subclinical mastitis were 60% dominated by S. epidermis.In the remaining 40% of samples, at least one of the dominant species was a species of the genus Streptococcus (often from the salivarius or mitis group), as well as S. aureus and S. epidermis [122].
Tuominem et al. did not detect differences in the composition of human milk microbiota related to HPV infection.However, it is important to note that this study included a small study group [123].Similar results were obtained by Bender et al. in relation to HIV [114].
Taking medications (including antibiotics), supplements, and chemotherapy may have a significant impact on the human milk microbiome.A link between the occurrence of microorganisms in human food and intrapartum exposure to antibiotics in milk a month after delivery has been demonstrated.Lower numbers of lactobacilli and Bifidobacterium spp.were observed [124].Subsequent studies show an increased presence of bacteria of the genus Acinetobacter, Xanthomonadacea, as well as a reduced presence of Bifidobacterium, Staphylococcus, and Eubacterium in the milk of women undergoing chemotherapy.Supplementation during pregnancy and lactation is extremely important for both the woman and the child.A positive correlation was found between vitamin C intake during pregnancy and the number of Staphylococcus spp., as well as the impact of maternal vitamin B1 and B2 intake on the milk microbiome [125].
The impact of a breastfeeding woman's diet on the human milk microbiome has not been thoroughly investigated.Few scientific studies suggest that diet affects not only the intestines but also the composition of human milk, including the microbiome.A correlation has been noted between the intake of saturated (SFA) and monounsaturated fatty acids (MUFA), carbohydrates, protein, and the occurrence of microorganisms in milk [126].Cortes et al. associated carbohydrate intake with the occurrence of Staphylococcus spp.and Bifidobacterium spp. in human milk, while the genus Streptococcus was associated with the intake of n-3 PUFA [EPA and docosapentaenoic acid (22:5ω-3)] [125].Scientists [104,120] believe that the mother's diet during pregnancy may have a greater impact on the human food microbiome compared to the diet during lactation.Williams [120] obtained dietary intake data and linked it to milk microbiome data obtained from 21 lactating women at the same time.Few associations were found between dietary intake variables and the relative abundance of bacteria in milk.However, when dietary and microbiome data are averaged over the entire observation period, a myriad of significant associations of phyla or phylum, respectively, with specific nutrients (e.g., negative association between Corynebacteria and SFA and MUFA) and macronutrients (e.g., reported carbohydrate intake and Firmicutes) can be found [120].In turn, Drago et al. showed no effect of diet on the microbiome of human milk [104].• 1 g increase in fiber content in cereals was associated with reduced incidence of Fusobacteria and Streptococcus and an increase in Acinebacterium; • trans fats showed a positive relationship with the occurrence of Staphylococcus and Gemella; • A 1 g increase in monounsaturated fat intake was associated with an increased incidence of Acinetobacter and Gemella; • negative associations were observed between the consumption of polyunsaturated fats and the incidence of Acinetobacter.

Microbiota Analysis Method Microbiota Diversity References
Lactation phase breastfeeding women MALDI-TOF-MS • total number of bacteria higher in colostrum than mature milk.Damaceno et al. [106] Mastitis women (10 healthy, mastitis) MALDI-TOF-MS • high absolute abundance of S. aureus in women with acute mastitis and S. epidermidis in women with subacute mastitis.

Jiménez et al. [33]
A type of breast pumping women 16S rRNA gene sequencing • higher content of Gemellaceae, Vogesella, and Nocardioides with manually expressed milk, higher relative abundance of Enterobacteriaceae and Pseudomonas in milk expressed by a breast pump.
Moossavi et al. [45] BMI women 16S rRNA gene sequencing • women with higher BMI had higher abundance of Granulicatella and lower relative abundance of Bacteroides.

Perinatal Factors
Scientific reports show that, from the neonatal period to infancy, the method of delivery is an important factor influencing the composition of the intestinal microbiota (Table 3).Moreover, research conducted on 596 full-term children showed that the method of delivery is the most important factor influencing the composition of children's intestinal microbiota [131].Newborns born vaginally acquire bacteria from the mother's birth canal, mainly species of the genus Lactobacillus and Prevotella.However, children born by cesarean section acquire bacteria that resemble the mother's skin microbiome, such as Staphylococcus spp.[132,133].Babies born by cesarean section have lower levels of anaerobic bacteria, such as Bacteroides spp.and Bifidobacterium spp.and are also colonized by such bacteria as Clostridium, Cutibacterium, and Corynebacterium [134].Cesarean section delivery has also been associated with lower abundance and diversity of the Actinobacteria and Bacteroidetes phyla, as well as greater abundance and diversity of the Firmicutes phylum from birth to 3 months of age [135].The conclusions of the systematic review seem interesting, indicating that the method of delivery is most important for the diversity and colonization pattern of the intestinal microbiome in the first three months of a child's life.However, the method of delivery has a smaller impact on the colonization and diversity of Bifidobacteria, Bacteroides, Clostridium, and Lactobacillus genera from 6 to 12 months of age [136].
Another important factor influencing a child's intestinal microbiome is the duration of pregnancy.Research analysis indicates that full-term children have a different composition of intestinal microbiota than premature babies [137].Research indicates that at all stages of lactation, a group of premature babies has significantly lower levels of Bifidobacterium spp.than a group of full-term babies [76].
Intestinal colonization in premature babies is influenced by many factors that cause disturbances in the intestinal ecosystem or dysbiosis-including necrotizing enterocolitis (NEC), which is the main cause of mortality in premature infants [138].Interesting studies on a group of 29 premature infants born between 28 and 32 weeks of pregnancy indicate a higher number of Clostridiates and a lower number of Enterobacterium among female infants compared to male infants [139].Subsequent studies conducted on premature babies show low species diversity.In this study, Bacilli, Proteobacteria, and Clostridium were the most abundant and accounted for 87%, and Actinobacteria and Bacteroidia 6.5% and 5.1%, respectively [140].The intestinal microbiome of premature infants consists mainly of bacteria of the genera Enterococcus, Staphylococcus, Streptococcus, Citrobacter, Enterobacter, Escherichia (mainly E. coli), Klebsiella, Raoultella, Serratia, and Shigella, and the Enterobacteriaceae order.There are also genera such as Bacteroides, Clostridium, and Veillonella.Bifidobacterium and Lactobacillus (intestinal bacteria), whose function is to protect the intestines against pathogens, are present in premature infants only two months after birth.Moreover, in prematurely born children, in the first six weeks there is a decrease in the number of bacteria of the genera Staphylococcus, Shigella, Escherichia, and Prevotella, and an increase in bacteria such as Enterococcus, Streptococcus, Enterobacteriaceae, and Veillonella.Studies also indicate that the occurrence of NEC is associated with a more frequent presence of Enterobacteriaceae, Clostridium spp., and coagulase-negative staphylococci (CNS).However, the presence of Enterococcus faecalis is associated with a reduced risk of NEC [141].Willoughby et al. indicated a relationship between the occurrence of NEC and the presence of bacteria such as E. coli, Klebsiella spp., Enterobacter spp., Clostridium butyricum, Clostridium perfringens, Salmonella spp., Pseudomonas aeruginosa, Clostridioides difficile, CNS, S. aureus, C. glabrata, coronavirus, enterovirus, and rotavirus [142].Another study conducted on a group of 369 premature infants showed that the presence of Klebsiella spp.and Clostridium spp. was strongly associated with an increased risk of NEC in children [143].
Postnatal exposure of the child to various environments during early intestinal development influences the colonization of the gastrointestinal tract and its immune system.Studies conducted from the 3rd to the 39th day of life on 58 premature babies staying in the Neonatal Intensive Care Unit (NICU) showed that approximately 92% of all bacteria in the stool are Proteobacteria (54%), Bacillus (19.3%), and Clostridia (18.4%) [144].Interesting research was carried out in the Intensive Care Unit (ICU), where samples of bedding, frames, and furniture were taken after cleaning twice with a 500 ppm free chlorine solution.The results of sample analysis showed the presence of bacteria such as Enterococcus spp., S. aureus, Klebsiella spp., Acinetobacter spp., P. aeruginosa, and other Enterobacteriaceae.These microorganisms also occur on the surface of the NICU and are the most common cause of hospital infections [145].Another study assessing 16 NICU room surfaces showed that the majority of microorganisms were associated with the skin-over 50% (Corynebacterium spp.)-mouth (Streptococcus spp.), or nose (Staphylococcus spp.).Interestingly, the floor in front of the infant isolation room had the highest microbial density compared to any other NICU environment [146].
According to the literature, the composition of the human milk microbiome is also influenced by parity.In their research, Kim et al. observed a greater presence of Staphylococcus spp.and Haemophilus spp. in bacterial samples from multiparous women compared to samples from primiparous women [147].
Scientific research shows that the vaginal microbiome changes with gestational age [148].Moreover, subsequent research analyzes suggest that when the microbiological balance in the vaginal ecosystem is disturbed, the risk of premature birth increases [135].Research conducted as part of the Human Microbiome Project (iHMP) of the National Institute of Health showed that premature birth was associated with low numbers of Lactobacillus spp.bacteria in the vagina, especially among African American women.These studies also showed a positive correlation of four taxa: S. amnii, BVAB1, Prevotella cluster 2, and TM7-H1-with the occurrence of preterm birth, which may be important in predicting the risk of preterm birth [149].La Rosa et al. observed that the intestinal microbiota of premature infants staying in a strictly controlled microbiological environment changes from Bacilli to Gammaproteobacterial to Clostridia.Moreover, when prematurely born children approach 33-36 weeks of post-conceptional age, their intestines are well colonized by anaerobes [144].
An analysis of intrapartum antibiotic therapy showed a delay in the maturation of microbial activity from 6 to 12 months after delivery [150].An increase in the number of Enterobacteria in infants treated with antibiotics and a reduction in the number of Bacteroides and Atopobium clusters in children born to mothers treated with antibiotics during pregnancy or lactation were observed [151].In their research, Hermansson et al. noticed the absence of Bifidobacterium spp.bacteria in the milk of women who received intrapartum antibiotic therapy [124].The results of this study may be important because reduced Bifidobacterium spp. in early infancy increases the risk of atopy and obesity [30].Intrapartum exposure to antibiotics also influences the development of NEC [135], as well as the occurrence of infantile colic [152].
The method of feeding plays an important role in shaping the microbiota of human milk.According to the literature, direct breastfeeding has a positive effect on the absorption of bacterial microbiota from the child's oral cavity, while feeding the child with expressed human milk increases the risk of acquiring bacteria from the environment [75].Moossavi et al. observed a significantly higher prevalence of Bifidobacterium spp. in directly breastfed children than in children fed with expressed human milk.Furthermore, Gemellaceae, Vogesella, and Nocardiosis were more abundant in direct breastfeeding, while Enterobacteriaceae and Pseudomonas spp.Were relatively more abundant in indirect breastfeeding [45].Many scientific studies have indicated differences in the composition of the intestinal microbiota between breastfed and formula-fed children.Through breastfeeding, the child acquires a large amount of Bifidobacterium spp., which constitutes approximately 90% of the total intestinal microbiome in the first year of the child's life [153].This is confirmed by subsequent studies indicating the predominance of Bifidobacteria in the feces of exclusively breastfed infants, while Enterococcus spp.and Clostridium spp.predominated in formula-fed children [28,154,155].In addition, children fed with formula milk acquire more microorganisms such as E. coli, C. difficile, Bacteroides, Firmicutes, and Lactobacilli [156,157].Zimmermann et al., reviewing the factors influencing the composition of the intestinal microbiome in the first year of life, indicate that in breastfed children, a greater number of Staphylococcus and Streptococcus species are observed, while in children fed with formula milk: Bacteroides, Clostridium, Enterobacteriaceae, Escherichia, Klebsiella, Enterococcus, and Lachnospiraceae, with slower colonization by Bifidobacterium spp.[141].Research conducted on a group of 28 premature infants indicates that a greater diversity of the intestinal microbiome and a significantly higher number of Clostridiales and Lactobacillus bacteria are observed in premature infants fed with mother's milk than in premature infants fed with modified milk and/or donor milk [139].However, Korpela et al. found that the intestinal microbiome in breastfed premature infants develops in four phases.The first phase peaks between 25 and 30 weeks of post-conceptional age with the dominance of Staphylococcus spp.The second phase is dominated by Enterococcus spp., and the peak occurs between 30 and 35 weeks of post-conceptional age; the third phase, Enterobacter spp., and the peak at 35 weeks.The highest number of Bifidobacterium spp. was observed in the fourth phase, recorded after the 30th week of post-conceptional age.The third phase, dominated by Enterococcus spp.bacteria, was observed only in extremely premature infants and seemed to delay the succession of microbiota [158].• in premature infants, the most numerous phylum of Bacilli, Proteobacteria, Clostridia (87%); • Actinobacteria and Bacteroidia 6.5% and 5.1%, respectively.

Grier et al. [140]
Duration of pregnancy • increase in the number of operational taxonomic units by 0.45 units/week-with staphylococci being the main group; • little represented bacteria of the Bifidobacterium genus.• increase in the number of Enterobacter in infants treated with antibiotics; • reduction in the number of Bacteroides and Atopobium clusters in children of mothers using antibiotic therapy.
Fallani et al. [151] Antibiotic therapy women 16S rRNA gene sequence • lack of Bifidobacterium spp.bacteria in the milk of women who received intrapartum antibiotic therapy.
Hermansson [124] Reproduction women (16 primiparous, multiparous) 16S rRNA gene sequence, PCR • higher numbers of Staphylococcus, Collinsella and Haemophilus in multiparous women.Kim et al. [147] Feeding method women PCR • higher numbers of Bifidobacterium spp. in children fed directly by breast milk compared to children fed with expressed mother's milk; • in children who are directly breastfed, a relatively higher abundance of Gemellaceae, Vogesella and Nocardioides; • in children fed with expressed breast milk, a relatively higher number of Enterobacteriaceae and Pseudomonas.
Moossavi [45] Feeding method children 16S rRNA gene sequence, PCR • higher numbers of E coli, C difficile, Bacteroides and Lactobacillus in children fed with formula milk.

Penders et al. [156]
Feeding method children 16S rRNA gene sequence • in the first 6 months of life, higher numbers of Bacteroidetes and Firmicutes in formula-fed children.Ho et al. [157] Feeding method dyads (mother-child) culture • in children fed with formula milk, lower numbers of Bifidobacterium and higher numbers of Clostridium and Enterobacteriaceae (E.coli); • in 4-month-old exclusively breastfed children, a higher abundance of taxa used as probiotics, such as L. johnsonii/L.gasseri,L. paracasei/L.casei and B. longum; • in 4-month-old children fed with formula milk, higher numbers of Clostridioides difficile, Granulicatella adiacens, Citrobacter spp., Enterobacter cloacae, and Bilophila wadsworthia.

Bäckhed et al. [155]
Feeding method premature babies (28 and weeks of pregnancy) 16S rRNA gene sequencing • significantly higher abundance of Clostridiales and Lactobacillales in premature babies fed with human milk compared to children fed with formula.

Cong et al. [139]
Feeding method premature babies 16S rRNA gene sequencing • the development of the microbiota took place in four phases, with the dominance of Staphylococcus, Enterococcus, Enterobacter and finally Bifidobacterium.

Composition of Human Milk and the Microbiome
There are several studies in the scientific literature explaining the relationship between the composition of human milk and the microbiome of human milk.One of the few studies at the moment is the analysis by Moosavi et al., in which the general profile of fatty acids in milk was significantly related to the composition of the milk microbiota.It has been shown that fatty acids [22:6n3 (docosahexaenoic acid), 22:5n3, 20:5n3, 17:0, 18:0] and oligosaccharides (fucosyl-lacto-N-hexaose, lacto-N-hexaose, lacto-N -fucopentaose I) were associated with microbiota diversity [45].Several significant associations were observed between individual HMOs and the microbiota.Among mothers using expressed milk, the prevalence of Bifidobacterium spp. was associated with a lower disialyl-lacto-N-hexaose content [45].Kumar et al. described the association of lipid profile with microbiota.In their study, monounsaturated fatty acids (MUFA) in triacylglycerols (TAG) were negatively correlated with Proteobacteria (r = −0.43,p < 0.05).Additionally, over 90% of women in the study were positively associated with MUFA and polyunsaturated fatty acids (PUFA) in TAG, while saturated fatty acids (SAFA) in TAG were negatively associated with Streptococ-cus and Acinetobacter genera.In turn, in the case of phospholipids, the genus Lactobacillus was negatively associated with the genus MUFA (r = −0.23,p = 0.04), and n-3 PUFA was negatively associated with the genus Bifidobacterium (r = −0.26,p = 0.03) [102].Opposite results suggesting a lack of relationship between the occurrence of oligosaccharides in milk and the food microbiome were obtained by Moossavi et al. [45].

Microbiota Modulation-During Pregnancy and after Childbirth
Scientific research shows that colonization of the intestines with microorganisms takes place at several stages.According to the literature, the first stage probably begins in the prenatal period and ends several months or even several years after birth.These reports question the dogma of the "sterile" fetus [135].Currently, the issue of intestinal colonization is still very controversial.There is no consensus among scientists dealing with microbiology and the prenatal/perinatal period.Numerous scientific studies have indicated the presence of microorganisms in fetal tissues [36,168,169], although the latest studies deny this [170].The results of microbiome analysis conducted using metagenomic shotgun sequencing techniques indicate the presence of commensal bacteria in the uterus, placenta, amniotic fluid, and meconium [46,[171][172][173][174][175][176][177].
The studies presented in Table 4 indicate that microbial exchange occurs from mother to fetus, confirming that the fetal environment is not sterile.The study results also indicate the importance of the mother's microbial environment for the infant's developing immune system [25].The mother's oral microbiota is an important prenatal factor influencing the development of the child's intestinal microbiome [171,178].The intestinal microbiome of newborns is also influenced by the duration of pregnancy and the method of delivery, which is described in detail in Section 4.3.
Yatsunenko et al. indicated that the microbiome of the gastrointestinal tract of children at birth is characterized by greater inter-individual variability but significantly less diversity than the microbiome of the gastrointestinal tract of adults [153].Firmicutes, Proteobacteria, and Actinobacteria are the dominant phyla in the neonatal gastrointestinal microbiome.However, the dominant phylum in the adult gastrointestinal microbiome is Bacteroidetes [184].Also interesting are the studies of Collado et al. [46], who discovered common features of the microbiota when assessing the amniotic, placental, and meconium microbiota in newborns born by cesarean section.The same study shows that the predominant phylum in the amniotic fluid and placenta is Proteobacteria, with Enterobacter, Escherichia, and Shigella being the predominant bacterial genus, followed by Cutibacterium.Much smaller amounts of these bacteria were detected in the colostrum, meconium, and feces of infants.The exact mechanism by which these microorganisms are transmitted from mother to fetus is currently unknown, but research shows significant similarities between placental and oral microbiomes [171].Therefore, it can be assumed that the colonization of microorganisms in the fetal gastrointestinal tract is shaped by the microbiome of the mother's oral cavity, which is the source of bacterial translocation to the placenta [171,178].The oral microbiota contains over 600 taxa in 13 phyla, including Chlamydiae, Synergistetes, Firmicutes, Actinobacteria, Bacteroidetes, Chloroflexi, Proteobacteria, Euryarchaeota, Fusobacteriia, Spirochaetes, and Tenericutes [185].Studies conducted on pregnant women and 6 weeks after delivery indicate that the diversity of the oral microbiota during pregnancy remains relatively stable.However, pathogenic hormonal fluctuations in pregnant women may affect the composition of the oral microbiome [186].In turn, oral microorganisms may be related to the intrauterine environment, which may interact with many adverse effects of pregnancy, such as premature birth or a deficit in the child's neurological development [187].
Many commensal bacteria occur in the placenta and uterus.Proteobacteria are dominant [171,174], but the most common single species is E. coli [187].A systematic review of 24 studies of the placental microbiome indicated that the most frequently identified genus was Lactobacillus [188], which was also the predominant genus in human milk [189].Lactobacillus spp., which has a protective function, was associated with a healthy vaginal and intestinal microbiome [190].In turn, the uterine microbiome contains not only Lactobacillus bacteria, but also Firmicutes, Bacteriodetes, Proteobacteria, and Actinobacteria [191].The presence of microorganisms in the prenatal period was also confirmed by tests conducted after delivery.Jiménez et al. showed that in the meconium of 21 healthy newborns, the dominant population are the genera Enterococcus and Staphylococcus [41,175], while at the phylum level the meconium microbiota is dominated by Firmicutes [175].Studies assessing the relationship between the meconium microbiome of 301 newborns and neonatal jaundice seem interesting, showing a relationship between a higher abundance of Bifidobacterium pseudolongum and a higher alpha diversity and a lower risk of jaundice in infants born by cesarean section.The same study showed that meconium consists mainly of bacteria of the genus Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and Bacteroidetes [180].In turn, Morais et al. showed that extremely premature infants born before 28 weeks of gestation had more Lactobacillus bacteria, which dominate the mother's vaginal microbiota, than infants born after 28 weeks of gestation, regardless of the mode of delivery.This confirms the hypothesis that maternal bacteria originating from the vagina and intestines play an important role in shaping the intestinal microbiota of newborns, and that the transfer of bacteria from mother to child is a controlled and time-bound process [182].Enterococcus aecium, Cutibacterium acnes, S. epidermidis, and Streptococcus sanguinis have been identified in the umbilical cord blood of healthy neonates born by cesarean section [177].The intestine of newborns is initially dominated by the genera Bifidobacterium, Veillonella, Streptococcus, Citrobacter, Escherichia, Bacteroides, and Clostridium, which are also abundant in the intestinal microbiota of adults [192], but eventually they are inhabited by the two dominant groups of anaerobic bacteria Firmicutes and Bacteroidetes [193].
Despite numerous scientific reports on fetal colonization, Kennedy et al.'s recent studies did not show evidence of the existence of a fetal microbiome, which calls into question previous findings.Kennedy et al. concluded that low microbial biomass and contaminants are responsible for erroneous prior fetal microbiome findings [170].
However, it should be remembered that during pregnancy, the mother's metabolism and microbiota adapt to the most optimal state for the developing fetus [194,195].According to the literature, the diversity of vaginal bacterial microbiota is decreasing, but the number of Lactobacillus bacteria is increasing [196].The intestinal microbiota of a pregnant woman also changes.The number of proteobacteria and actinomycetes increases, but the number of bacteria that produce butyrate decreases.There is also a reduction in the diversity of bacteria in the gastrointestinal tract [197].In turn, during natural childbirth, the baby passing through the birth canal is exposed to a variety of microorganisms that come from the birth canal and the mother's intestines, as well as from the hospital environment.The formation of the composition of the intestinal microbiota of newborns is a dynamic and slow process.Lactobacillus and Enterobacteria, as well as other facultative anaerobes, colonize the intestines first, and obligate bacteria, such as Bifidobacteria and Bacteroides, colonize the intestine within a week of birth.Scientific research has shown that the development of a child's intestinal microbiota is not stable.In the first year after birth, Bifidobacteria predominate, but genera such as Fusobacterium and Ruminococcus Enterococcus, Staphylococcus, Streptococcus and Cutibacterium have also been identified [177].The fundamental factor shaping the human intestinal microbiota is diet [198].The World Health Organization recommends introducing complementary foods after the baby is 6 months old.The introduction of solid foods into a child's diet supplements the intestinal microbiota, which becomes more diverse and similar to the intestinal microbiota of an adult [199,200].The latest scientific research highlights the important role that microorganisms play in early life in the maturation and development of infants.Creating a good environment for the growth of various types of microorganisms can influence the proper development of the intestinal microbiota of infants and young children [201].According to studies, important microbiota include Saccharomyces boulardii, LGG (Lactobacillus rhamnosus), Lactobacillus reuteri, and Bifidobacterium lactis (BB-12).It cannot also be ignored that probiotics have a significant impact on the development of intestinal microbiota in infants and young children, and also have the ability to prevent diseases such as diarrhea, infections [202], allergies [203], and neonatal colic [204].

Does the Composition of Human Milk Influence the Formation of a Newborn's Microbiota?
According to scientific reports, colonization of the gastrointestinal tract in children is significantly influenced by the microbiome of human milk.Therefore, it is important to ensure that, especially in the first 100 days of the child's life, there are no disturbances of the human milk microbiome, which could consequently contribute to the destruction of the microbiological balance of the child's intestines [75].The composition of the human milk microbiome affects the baby's health in two ways: it facilitates digestive processes and also promotes intestinal immune homeostasis [26].Bacterial diversity plays a key role in maintaining the immune balance in both children and adults [205].In particular, bacteria derived from human milk provide early antigenic stimuli that have a beneficial effect on the maturation of the intestinal immune system [26].Research indicates that approximately 25-30% of children's intestinal microbiota come from human milk [43].In exclusively breastfed children, a lower diversity but higher number of Lactobacillus spp.B. breve and B. bifidum bacteria were observed compared to formula-fed children.In turn, cessation of breastfeeding results in faster maturation of the intestinal microbiome, as evidenced by the Firmicutes phylum [206].Primary colonization of Lactobacillus spp.and Bifidobacterium spp.results in a more acidic intestinal environment with high concentrations of short-chain fatty acids, which are reproduced by bacterial fermentation of human milk oligosaccharides.Therefore, it is important to remember that human milk controls the development of the newborn's intestinal microbiota indirectly (transfer of prebiotics and promoting the growth of specific bacterial species such as Bifidobacterium spp.) as well as directly (vertical transfer of pioneer bacterial species) [26].
Determining the correct profile of intestinal bacteria is important in preventing dysbiosis, as well as minimizing its impact on health [207].In prematurely born children, NEC is a consequence of an enhanced and devastating inflammatory response to intestinal dysbiosis, resulting in tissue damage and impaired intestinal barrier integrity [208].Microbial dysbiosis may be a risk factor for the development of diseases not only in the first months of life but also later in childhood.In particular, dysbiosis of the respiratory and digestive tracts may cause the development of asthma and obesity [209].Moreover, metabolites that are recreated by microorganisms moving from the damaged intestinal barrier can affect several organs, causing systemic metabolic inflammation [63].Therefore, both diseases of the neonatal period, such as NEC, and diseases that begin later in life, such as asthma and obesity, are clinical symptoms of early microbiota dysbiosis [210].
The impact of breastfeeding on shaping the intestinal microbiota of children is the best researched and documented.However, it should be remembered that breastfeeding also has a significant impact on shaping the microbiota of the oral cavity, nasopharynx, and respiratory tract.This is confirmed by research by Holgerson et al., who showed differences in the oral microbiota between 3-month-old breastfed infants and 3-month-old infants fed formula milk.The presence of Lactobacillus species was found in 27.8% of exclusively and partially breastfed infants, but not in formula-fed infants [211].Subsequent studies indicate that the Firmicutes phylum dominated the oral bacterial environment in both groups (breastfed vs. formula-fed infants).A higher prevalence of Prevotella and the Bacteroidetes phylum was observed in the oral cavity of formula-fed infants compared to breastfed infants.However, a higher frequency of Proteobacteria and Actinobacteria species was found in the oral microbiota of breastfed children [40].Analysis of the oral microbiota of infants and colostrum collected from pregnant women before delivery revealed common types of bacteria, with Streptococcus spp.and Staphylococcus spp.being the most abundant.The presence of typical oral microbiota in colostrum analyzed before birth, i.e., before the contact of the mammary gland with the newborn, suggests that breastfeeding is one of the first microbiological sources for the formation of the child's oral microbiota [39].In turn, a study aimed at comparing the microbiome of human milk and the microbiome of oral and fecal samples of healthy breastfeeding women and their infants showed that variability of the human milk microbiome may have an impact on the microbiome of the infants' gastrointestinal tract.Additionally, the most abundant genera in both mother and infant oral samples were the genera Streptococcus, Staphylococcus, Gemella, Rothia, and Veillonella [212].Dzidic et al. showed that shorter breastfeeding habits during the first two years of a child's life were associated with a distinct oral bacterial composition later.The results of these studies also describe the development of oral microbiota as ecological succession.Altered bacterial colonization patterns during the first year of life may have long-lasting effects on both the oral and systemic health of children.Changing the pattern of bacterial colonization during the first year of life can have long-lasting effects on both the oral and systemic health of the child [213].
Scientific research shows that breastfeeding reduces the risk of respiratory infections in the neonatal and infancy period.It has been proven that breastfed children get sick less frequently than formula-fed children, and that the duration of infection is shorter and its severity is weaker.Biesbroek et al., analyzing the nasopharynx microbiota in 6-weekold infants, observed a significant difference in the microbiological composition between children fed with formula and breastfed children [214].Formula-fed children had lower numbers of lactic acid bacteria and increased numbers of Staphylococcus spp.and anaerobic bacteria, such as Prevotella and Veillonella species.In turn, the respiratory microbiota of breastfed infants showed low abundance of the following genera: Veillonella, Prevotella, Streptococcus, Rothia, Gemella, and Granulicatella compared to children who were fed with formula milk.Interestingly, the occurrence of the Dolosigranulum genus was reported to be inversely related to the symptoms of wheezing and the number of previous respiratory infections.However, based on the research of Biesbroek et al., it can be concluded that at the age of 6 months, the connection between breastfeeding and the composition of the nasopharyngeal microbiota disappears [214,215].

Microbiome Assessment Methods
So far, research on the assessment of the presence of microorganisms in human milk has been conducted using classic microbiological methods, including cultures and identification using biochemical and immunological methods.The development of molecular biology techniques allowed the use of new methods, including mainly the PCR reaction with its variants and sequencing (Figure 4).The use of new techniques has allowed for the confirmation of a rich and diverse microbial community (including non-cultured ones) in human milk samples [50,78,216].So far, it has been shown that culture methods can estimate the number of bacteria at the level of 10 2 to 10 4 CFU/mL, while the use of cultureindependent methods may reveal a bacterial titer at the level of 10 4 to 10 5 CFU/mL, which may indicate the presence of dead bacteria and non-culturable species [49].The first study of the milk microbiome using pyrosequencing showed that lactic acid bacterial communities are generally complex [50].
Using breeding methods, Jost et al. showed that the average number of live bacteria in human milk was low and was at the level of 3 log CFU/mL [78].This is consistent with the results presented, among others, by Perez et al. and Heikkila et al. [51].In turn, Solis et al. showed that the average bacterial values in the milk of healthy women were 3-5 log CFU/mL [68].Discrepancies between studies may result from the microbiological media used, culture conditions, and other factors influencing the formation of the milk microbiome, including geographic location.
Both breeding and molecular biology methods have certain limitations.Obtaining different results between researchers may result from different protocols for sample collection and preservation (e.g., aseptic methods, time of day, breast cleaning methods, collecting samples before or after feeding, manual expression or expression with a breast pump) and, at a later stage, DNA extraction (e.g., from whole or skimmed milk, use of commercial kits, enzymatic lysis), selection of specific primers, and sequencing platforms [50,78,216].
It is desirable that standardized protocols for sample collection and validation of the methods used be developed in the future.Due to the composition of cell walls, DNA isolation methods, and the number of reference gene copies, the total bacterial count may be overestimated or underestimated [33].
Sequencing is unable to distinguish live from dead microbes or from cell-free DNA, so viability dyes, such as propidium monoazide, are used [217].Another limitation is that it is impossible to obtain quantitative results using this method, and, at best, only semiquantitative data can be obtained.
As recommended by Jimenez et al., testing of human milk samples should be preceded by blind control sequencing.If such control is not carried out, the results obtained should be analyzed with caution [33].In turn, Soto et al., comparing the results from breeding techniques and sequencing, showed high consistency of the results obtained, both in relation to the genus and species [71].

Perspectives
Information about microorganisms in human milk has been limited so far.There has been an increase in research on this topic in recent years.However, the origin of these populations in milk is not fully understood and remains the subject of much debate.Moreover, the biological role of neonatal health in the short and long term has not yet been determined.The fundamental task for scientists working with human milk is to define a healthy microbiota and prevent microbial dysbiosis, which is crucial in the first months of a child's life and also influences the development of chronic diseases, such as obesity and asthma.The methods of collecting milk samples and assessing Sequencing is unable to distinguish live from dead microbes or from cell-free DNA, so viability dyes, such as propidium monoazide, are used [217].Another limitation is that it is impossible to obtain quantitative results using this method, and, at best, only semi-quantitative data can be obtained.
As recommended by Jimenez et al., testing of human milk samples should be preceded by blind control sequencing.If such control is not carried out, the results obtained should be analyzed with caution [33].In turn, Soto et al., comparing the results from breeding techniques and sequencing, showed high consistency of the results obtained, both in relation to the genus and species [71].

Perspectives
Information about microorganisms in human milk has been limited so far.There has been an increase in research on this topic in recent years.However, the origin of these populations in milk is not fully understood and remains the subject of much debate.Moreover, the biological role of neonatal health in the short and long term has not yet been determined.The fundamental task for scientists working with human milk is to define a healthy microbiota and prevent microbial dysbiosis, which is crucial in the first months of a child's life and also influences the development of chronic diseases, such as obesity and asthma.The methods of collecting milk samples and assessing microorganisms in milk should also be discussed.The topic of the milk microbiome is still relevant and requires research in various aspects by scientists around the world.

Figure 2 .
Figure 2. Core human milk microbiome and the dominant species in colostrum and mature milk.

Figure 2 .
Figure 2. Core human milk microbiome and the dominant species in colostrum and mature milk.
58 children (n = 15 children born < 26 weeks of gestation; n = 20, 26-28 weeks of pregnancy; n = 23, >28 weeks of pregnancy 16S rRNA gene pyrosequencing • the intestinal microbiota of premature infants changes from Bacilli to Gammaproteobacteria to Clostridia.27 children born between 24 and 32 weeks of pregnancy; n = 13 children born between 37 and 41 weeks of pregnancy) 16S rRNA gene sequence • in premature babies in the first months of life, a lower number of the Bacteroidaceae family and a higher number of Lactobacillaceae compared to full-term children.Arboleya et al. [162] Duration of pregnancy 41 children (n = 21 children born between 30 and 35 weeks of pregnancy; n = 20 children born between 38 and 41 weeks of pregnancy) qPCR • in premature infants, increased number of facultative anaerobes Enterobacteriaceae, Enterococcaceae and Lactobacillus (including Weissella); • in premature babies, reduced numbers of anaerobic bacteria, including Bifidobacterium, Bacteroides, and Atopobium,

Figure 4 .
Figure 4. Methods for assessing the human milk microbiome, including advantages and disadvantages.

Figure 4 .
Figure 4. Methods for assessing the human milk microbiome, including advantages and disadvantages.

Table 1 .
Diversity of the human milk microbiome depending on geographical location.
[68]gher abundance of Proteobacteria in South Africa women compared to women from Finland, Spain, and China; • Bifidobacteriaceae was found in women from South Africa, while no Bifidobacteriaceae was found in women from Finland, Spain and China.•higherabundance of Streptococcus spp. in Chinese women compared to women from Finland, Spain and South Africa;• Enterococcaceae was found in samples from women from South Africa, Finland, and Spain, while no Enterococcaceae was found in women from China.• higher abundance of Bacteroidetes in Spanish women (natural childbirth) compared to women from South America and China;• higher abundance of Firmicutes among women from Finland compared to women from South Africa and China; • higher abundance of Cutibacterium and Pseudomonas spp. in Spanish women compared to women from South Africa and China.•colostrumandmaturemilkshowed high bacterial counts: > 200 generations/sample; • higher relative abundance of anaerobic bacteria in mature milk compared to colostrum; • predominance of Abiotrophia and Alloiococcus in colostrum and Parabacteroides in mature milk.• the most frequently isolated species of the Staphylococcus genus was S. epidermidis, and of the Streptococcus genus-S.salivarius;•5% of all isolates belonged to the genus Lactobacillus, and another 5% were Bifidobacterium spp.;• the most frequently isolated LAB was Lactobacillus gasseri.Solís et al.[68]

Table 2 .
Diversity of the breast milk microbiome depending on maternal factors.

Table 3 .
Diversity of the gut microbiome of newborns and infants depending on perinatal factors.• in newborns born by cesarean section, a higher number of enterobacteria, i.e., Klebsiella and Enterobacter;• Bacteroides colonization delayed up to 1 year in children born by cesarean section.

Table 3 .
Cont. the most numerous phylum Proteobacteria; • current shift patterns: increase in Clostridium and Bacteroides and decrease over time in early life; • higher numbers of Clostridiates and lower numbers of Enterobacterium in girls than in boys. •

Table 4 .
Diversity of the microbiome from the placenta, amniotic fluid, meconium, and cord blood.Firmicutes phylum, the dominant genera are Streptococcus, Lactobacillus, and Veillonella;• in the Proteobacteria phylum, the dominant genera are Pseudomonas, Haemophilus, and Acinetobacter.Candida fungi have been identified: C. albicans, C. tropicalis, and C. glabrata • 30 species of bacteria were identified; Escherichia coli belonging to the Enterobacteriaceae family constituted 57.71% of the isolated strains, and Enterococcus faecalis belonging to the Enterococcaceae family constituted 21.03% of the isolated strains.