Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve.

Increasing evidence supports the importance of the breast milk microbiome in seeding the infant gut. However, the origin of bacteria in milk and the process of milk microbe-mediated seeding of infant intestine need further elucidation. Presumed sources of bacteria in milk include locations of mother-infant and mother-environment interactions. We investigate the role of mother-infant interaction on breast milk microbes. Shotgun metagenomics and 16S rRNA gene sequencing identified milk microbes of mother-infant pairs in breastfed infants and in infants that have never latched. Although breast milk has low overall biomass, milk microbes play an important role in seeding the infant gut. Breast milk bacteria were largely comprised of Staphylococcus, Streptococcus, Acinetobacter, and Enterobacter primarily derived from maternal areolar skin and infant oral sites in breastfeeding pairs. This suggests that the process of breastfeeding is a potentially important mechanism for propagation of breast milk microbes through retrograde flux via infant oral and areolar skin contact. In one infant delivered via Caesarian section, a distinct strain of Bifidobacteria breve was identified in maternal rectum, breast milk and the infant's stool potentially suggesting direct transmission. This may support the existence of microbial translocation of this anaerobic bacteria via the enteromammary pathway in humans, where maternal bacteria translocate across the maternal gut and are transferred to the mammary glands. Modulating sources of human milk microbiome seeding potentially imply opportunities to ultimately influence the development of the infant microbiome and health.

Introduction

The complex interplay between the microbiome, maternal immune constituents and infant gut colonization is of great importance to the development of the human microbiome, however, the sources of microbes in human milk still require further elucidation. Both culture and non-culture methods have identified aerobic and anaerobic bacterial species in milk, including strict anaerobes typically compartmentalized in the gut [1-6]. Precolostrum, prior to labor, contains bacterial species similar to milk after labor[6-8]. The same microbes have been found in both milk and feces of mother-infant pairs[5]. Human milk plays an important role in establishing the infant gut microbiome, serving as a source of lactic acid-producing bacteria and human milk oligosaccharides for the infant gut[9, 10]. Similar to murine models[11], human milk and its microbes facilitate differentiation of the neonatal intestinal epithelium, development of the gut associated lymphoid tissue and maturation of the neonatal immune system [12].

Proposed sources for the bacteria in human milk include skin and areolar bacteria, the environment, and infant’s oral microbiota through retrograde flow that occurs during nursing[6, 8, 13]. Alterations in the bacterial composition of human milk have been associated with maternal BMI, weight gain, hormones, lactation stage, gestational age, and mode of delivery[13, 14]. Although controversial, the presence of an enteromammary pathway, whereby bacteria, assisted by dendritic cells, translocate across the maternal intestinal mucosa and are delivered to the lactating mammary gland, has been proposed as one source of the bacteria including anaerobes in pre-colostrum and milk[8, 15]. There is some supportive evidence that maternal ingestion of probiotics increases breast milk levels of these microbes[16-18]. If this pathway proves to exists in humans, this suggests that modulation of maternal gut flora may directly impact infant health [15]. While murine [11, 19] and bovine studies [20, 21] suggest that bacteria enter milk from an enteromammary pathway, this is challenging to prove in humans and has been the subject of debate.

Disentangling the contributions of potential sources of bacteria in breast milk is difficult. We sought to assess breastfeeding and potential retrograde flow of bacteria from the infant’s oral cavity by performing 16S rRNA gene sequencing on samples from two groups of mother-infant pairs, one in which infants latched onto their mother’s breast and a second group of infants that never latched. Furthermore, we investigate the potential role of an enteromammary pathway to the human milk microbiome by performing shotgun metagenomic sequencing in an infant born via Caesarian section. We found that the process of breastfeeding is a potentially important mechanism for propagation of breast milk microbes through retrograde flux via infant oral and areolar skin contact. Our data also implicates a connection between Bifidobacteria breve in maternal gut and breast milk suggesting that intestinally-derived bacteria may translocate to the mammary gland and colonize the infant intestine.

Materials and methods

A subset of mother-infant pairs were selected from a larger cohort who delivered in Los Angeles, California from 2010 to 2014. The Institutional Review Board of Children’s Hospital of Los Angeles approved the study and written consent was obtained. Fifteen of the mother-infant pairs latched for breastfeeding and 5 infants who had never latched were selected for comparison. Samples collected included expressed milk, maternal areolar skin swabs, and infant stool samples as previously described [22]. Swab samples were also obtained from the mother’s oral mucosa, vagina, and rectum and the infant’s buccal mucosa. To capture what the baby was actually exposed to, the study coordinator, wearing standard laboratory gloves, collected one swab (Copan, Murrieta, California, USA) from each maternal areola after the mother performed her typical cleaning, but before the baby latched. After collection, samples were transported on ice, and then were either placed in Stool DNA Stabilizer buffer (Stratec, Berlin, Germany) or frozen ‘neat’ within 4 hours of collection and stored at -80°C.

DNA extraction and purification was performed on frozen human milk samples, areolar skin samples, stool samples, and swabs obtained from the oral mucosa, vagina, and rectum as previously described[22]. Quantitative PCR (qPCR) was used to determine the copies of 16S and GAPDH genes per ng of total DNA extracted from each human milk sample. 16S targeting primers 515F (GTG YCA GCM GCC GCG GTA A) and 806R (GGA CTA CNV GGG TWT CTA AT) were designed based on Caporaso et al[23] and acquired from Eurofins Genomics (Louisville, KY). GAPDH primers GAPDH-for (ACC ACA GTC CAT GCC ATC AC) and GAPDH-rev (TCC ACC ACC CTG TTG CTG TA) were acquired from IDT (Skokie, Illinois) as ready-made primers. Quantitation for 16S and GAPDH targets were performed separately in qPCR reactions containing 1x SSO Advanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA), and 0.5 uM of each paired primer and approximately 1 ng of template DNA. qPCR thermocycling was carried out using a Bio-Rad CFX96 instrument with the following conditions: GAPDH, 98C hold for 2 min followed by 40 cycles of 98C for 20 sec and 60.5C for 40 sec; 16S, 98C hold for 2 min followed by 40 cycles of 98C for 20 sec and 61.C for 40 sec. Standards for GAPDH were obtained by 10-fold serial dilutions of DNA extracted from human T cells and standards for 16S DNA were prepared as described previously[22]. The samples and standards were analyzed in triplicate using the CFx Maestro program (Bio-Rad) and results are reported as the mean log copies/ng of total DNA.

For all 20 subjects, the V4 region of the 16S rRNA gene was amplified and sequenced as previously described[22, 24, 25]. DNA amplicon concentrations were then quantified on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Pooled libraries were sequenced on an Illumina MiSeq instrument using 2x150bp v2 chemistry [25]. DADA2 version 1.4 was used for error correction, sequence inference, and chimera filtering with default settings. Taxonomic classification was performed using the RDP naïve Bayesian classifier. Contaminant sequence variants were identified as those with at least 10% of their abundance derived from negative control samples and were excluded from all subsequent analyses as previously described[26]. Diversity, ordination, and permutational multivariate analysis of variance (PERMANOVA) analyses were performed using the ‘phyloseq’ (version 1.22.3) and vegan (version 2.5–2) R packages. PERMANOVA assesses overall microbial variation by measuring the fraction of variance that can be explained by each covariate. Zero-inflated negative binomial (ZINB) regression models were used to test for differential abundance of specific bacterial taxa using rarefied sequence counts as the outcome and clinical covariates as the independent variable. Infant age in days was included as a covariate in all models to account for differences in microbial composition by age. The Benjamini-Hochberg FDR method was used to control for multiple hypotheses and results with an adjusted p-value less than 0.05 were accepted as significant. Source tracking analysis to help determine site contribution to breast milk and infant stool was performed using SourceTracker version 1.0.0 with default parameters and the amplicon sequence variant (ASV) table as input.

Shotgun metagenomic sequencing was performed as previously described[2] on 6 subjects in the latched cohort. Metagenomic libraries were constructed from the previously extracted DNA using the Illumina Nextera XT DNA library preparation kit following manufacturer’s instructions. Sequencing was performed on a NextSeq500 platform to a target depth of 5 million reads per sample. Adapter trimming and quality filtering were performed using trim galore, host sequences were removed using kneadData, and taxonomic classification was performed with Kraken (v0.15-beta). ConStrains was used to perform strain-level analysis with parameters ‘min-coverage 5’.

Results

Fifteen mother-infant pairs where the infant latched during breastfeeding and 5 mother-infant pairs whose mothers expressed breast milk but the infants did not latch for medical reasons were included (Table 1). Maternal age and length of pregnancy were similar between the two groups. However, more mother-infant pairs in the latched group were delivered vaginally (53%) whereas the majority (80%) of the non-latched group underwent a non-elective Cesarean section. More of these never-latched infants (40%) and mothers (80%) received antibiotics than their latched counterparts. In the never latched cohort, 2 maternal-infant pairs received both maternal intrapartum and infant postpartum antibiotics. Their samples were collected during the first 3 weeks of life. The other 2 never latched maternal-infant pairs had maternal intrapartum antibiotics only. They were sampled in the first 3 days of life. In the latched cohort, one mother-infant pair received both maternal intrapartum and infant postpartum antibiotics; the infant’s sample was collected at DOL 19. Six additional mothers in the latched group received antibiotics during delivery only and were sampled from DOL 3 to 55.

Table 1

Clinical characteristics of mothers and their infants (n = 20 mother-infant pairs).

Demographics of mother-infant pairs
	Latched (n = 15)	Never-latched (n = 5)
Maternal age (years)	31 (17–38)	25 (23–46)
Length of pregnancy (weeks)	39 (33–41)	38 (34–41)
Mode of delivery
Vaginal (%)	8 (53.3%)	1 (20%)
Elective Cesarean (%)	5 (33.3%)	0 (0%)
Non-elective Cesarean (%)	2 (13.3%)	4 (80%)
Maternal antibiotic treatment
Before deliverya	0 (0%)	0 (0%)
During deliveryb	7 (46.7%)	4 (80%)
After delivery	0 (0%)	0 (0%)
No antibiotic treatment	8 (53.3%)	1 (20%)
Infant gender (male:female)	7:8	4:1
Infant age (days)	22 (3–111)	5 (1–20)
Ethnicity
Hispanic	10 (66.7%)	3 (60%)
Caucasian	2 (13.3%)	1 (20%)
Asian	3 (20%)	0 (0%)
African American	0 (0%)	1 (20%)
Feeding
Exclusive breast milk	6 (40%)	0 (0%)
Mixed (formula + breast milk)	9 (60%)	3 (60%)
Nothing by mouth	0 (0%)	2 (20%)
Infant antibiotic treatment	1 (6.7%)	2 (40%)

Data are shown as median, range, or percentage.

aDuring pregnancy until 48 hours before delivery.

bDuring the 48 hours before delivery and in labor.

Of the 20 mother-infant pairs, 15 pairs (13 latched and 2 never-latched) were included in the final analysis. Five pairs were eliminated due to insufficient quantity of qPCR-recovered milk bacteria or DNA and were not true positives by qPCR (1.69–5.22 log 16S V4 copies/ng DNA). Of the included never-latched pairs, 1 subject had 4 different milk samples longitudinally collected within the first two weeks of life which were analyzed individually. In the breastfed group, breast milk bacteria were largely comprised of Staphylococcus, Streptococcus, Acinetobacter, and Enterobacter which were primarily derived from areolar skin and infant oral sites according to SourceTracker analysis (Table 2). Notably, the two mothers with never-latched infants showed different compositions with pure Staphylococcus in one and Staphylococcus, Finegoldia and Corynebacterium in the other (Fig 1A).

Table 2

SourceTracker analysis of the potential sources of the breast milk microbiome.

Sourcea	Meanb	Medianb	Standard Deviationb	Minimumb	Maximumb
Maternal areolar skin	0.45949	0.28783	0.39404	0.00397	0.99885
Infant oral	0.25818	0.08122	0.34048	0	0.93847
Maternal oral	0.01410	0	0.03555	0	0.15192
Maternal rectum	0.00002	0	0.00008	0	0.00037
Maternal vagina	0.00162	0	0.00691	0	0.03242
Unknown	0.26659	0.02206	0.37471	0	0.92936

aPercentages of the breast milk microbiome is inferred to come from each of these sources: maternal areolar skin, oral, rectum, vagina and infant oral cavity.

bMean, median, standard deviation, minimum, and maximum refer to the distribution of these percentages as it is calculated independently for each mother-infant pair.

Fig 1

Microbiome composition of human milk samples.

(A) Infant age (days) at time of sampling, relative abundance, maternal antibiotics in the 14 days prior to sampling, mode of delivery, and Shannon diversity of human milk samples from mothers with infants who either have latched or never latched. Samples from the same mother collected on different days are grouped. Milk from mothers who never had their infants latched were dominated by Staphylococcus in one and Staphylococcus, Finegoldia and Corynebacterium in the other. Note the absence of Streptococcus and lower overall diversity of never-latched samples. In contrast, samples from mothers with latched infants, also born via Caesarian section in the first 10 days of life (n = 5), contained Streptococcus, Acinetobacter, and Enterobacter in addition to Staphylococcus. (B) Relative abundance of genus Bifidobacterium by targeted 16S rRNA gene sequencing (left) and shotgun metagenomics (right) in a single milk sample (arrow) shown in Panel A. Bifidobacterium breve appears to be selectively cultivated in the mother’s milk and then makes up the majority of her infant's early gut microbiome.

In a sub-analysis of the latched samples, PERMANOVA identified exclusive breastfeeding as a significant driver of overall microbial variation (R2 = 0.028, p<0.001). No significant differences in diversity or relative abundance of specific bacterial taxa were noted by exclusive breastfeeding, delivery, or sex. Intriguingly, the genus Bifidobacterium on 16S rRNA sequencing was found in the breast milk, infant stool, and maternal rectal samples from a single mother-infant pair with Caesarian delivery. We utilized shotgun metagenomics to further resolve the strain identity of this shared bifidobacteria. Species-level analysis showed Bifidobacterium breve to be only a minor component of the maternal gut community (0.07% relative abundance) but a significantly larger portion of the breast milk and infant gut microbiomes (28.44% and 67.7% relative abundance, respectively) (Fig 1B). Strain-level mutational profiles also revealed a distinct strain of Bifidobacterium breve to be common across these three samples from the same mother-infant pair.

Discussion

The process of breast feeding plays a critical role in development of the infant gut microbiome. The initial seeding of the infant gut in the first few months of life is necessary for infant immune development and overall health[27-31] with breastfeeding exclusivity and percentage critically influencing the infant gut microbiome[22, 31]. In our analysis, the breast milk and infant microbiomes are seeded through multiple pathways, though primarily from areolar skin and infant oral sites. Additionally, a single mother-infant breastfeeding pair provide intriguing evidence for an enteromammary pathway contributing the same strain of Bifidobacterium breve found in maternal intestine and human milk as well as her infant’s gut. This infant was delivered via Caesarian section limiting the possibility of infant colonization during delivery. Furthermore, even though Bifidobacterium breve constituted less than 1% of the maternal rectal sample, it made up 28% of the maternal milk sample. This single species of bacteria then composed 68% of the infant’s gut microbiome.

There is increasing evidence of transfer of anaerobic bifidobacteria from maternal intestine to breast milk then colonizing and expanding in infant gut[5]. Bifidobacteria are amongst the first bacteria to colonize the infant intestine and are associated with decreases in the risk of obesity, asthma, atopy, and all-cause mortality from necrotizing enterocolitis in pre-term infants[27, 32, 33]. Given the importance of bifidobacteria in infant health, it is logical for mothers to selectively enrich and support colonization by this bacterial population.

Milk ducts are bidirectional channels [34] so it is likely that bacteria from skin and the infant oral cavity populate human milk. Furthermore, there is recent support that strains of bacteria found in precolostrum may have a significant impact in the initial establishment of the infant oral microbiota [6]. Our analysis is also suggestive of the role of retrograde oral seeding of bacteria into maternal milk from the act of infant suckling. Both mother-infant pairs with sufficient data where the infant never latched had a predominance of skin flora consisting mostly of Staphylococcus and some Corynebacterium with a notable absence of Streptococcus. In contrast, most of the milk samples from latching pairs had at least some and often a majority of Streptococcus present in their milk samples. Latched samples also had a greater overall diversity including Acinetobacter, Enterobacter, Veillonella, and Haemophilus in addition to the Staphylococcus, Streptococcus and Corynebacterium, consistent with previous studies[13]. However, with only 2 of the never-latched mothers having sufficient bacteria present in their milk for analysis, our data are insufficient to draw any definitive conclusions about the role of latching on milk microbe composition.

Our study is limited by the small sample size, the range of gestational ages of the infants at birth, the range of ages at sample collection, and by the inability to show directionality of bacterial transfer. Some microbes found on areolar skin are also present on the mucosal surfaces of the gastrointestinal tract[8] and our methods do not determine the source of these microbes in the breast milk. Prior investigations have demonstrated an enteromammary pathway in animals [8, 15] and a viable strain of Bifidobacterium breve in maternal faeces, breast milk and neonatal faeces in vaginally delivered infants [5]. Although our report suggest evidence for an enteromammary pathway by finding a single strain of Bifidobacterium breve in maternal rectum, breastmilk and infant stool, there is a possibility that maternal fecal microbes can be spread by the mother herself to the skin and breast although this is less likely in an infant delivered via Caesarian section. More definitive evidence is required to support the role of an enteromammary pathway in humans for translocating critical microbial communities to the breast milk compartment and eventually seeding the infant gut via breastfeeding. Our findings need to be investigated with larger cohorts and molecular-based surveys or culture-based analyses to validate the shotgun metagenomics data.

In conclusion, our data suggests that the process of breastfeeding and interaction between areolar skin and infant oral cavity are potentially critical for seeding the milk microbiome. Furthermore, our report provides intriguing evidence suggestive of an enteromammary pathway in humans with transfer of a single strain of Bifidobacterium breve in maternal intestine, breastmilk and infant stool in an infant delivered via Caesarian section. These sources of milk microbiome seeding, if verified in larger studies, may support opportunities to modulate bacteria found in human breast milk and ultimately development of the infant microbiome.

11 Aug 2019

PONE-D-19-16931

Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve

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Reviewer #1: The manuscript describes differences in human milk microbiota composition of mothers whose infants latched and those that had never latched. Data are also presented which suggest vertical transmission of a particular species of Bifidobacterium from the maternal gut to the infant gut by way of human milk. While some of the data are interesting, they seem incomplete. The differences in terms of gestational age and age when samples were collected across infants included in the study are also concerning.

Line 63-64: need citations

Line 63-65: Line is confusing. The existence of an enteromammary pathway is independent of seeding the infant gut. The enteromammary pathway is from the maternal gut to the mammary gland. It sounds as though you mean that vertical transmission is difficult to prove

Line 75: check the formatting of you bacterial names throughout the paper. This should either be bifidobacteria or Bifidobacterium breve.

Lines 85-87: The reference provided doesn’t offer much about information about collection processes, and there isn’t enough provided in the manuscript. Was the breast cleaned in any way before collection? How many swabs were used for each sample, and what type of swabs were used? Did mothers wear sterile gloves? Were the swabs that were not frozen immediately refrigerated?

Line 126: No SourceTracker results are presented in the manuscript. This does not need to be included in the methods if no results are presented. If you do choose to include it, what type of data did you put into the program – an OTU table or rarefied sequences?

Line 144: How close to sample collection did the infants receive antibiotics?

Table 1: Multiple of these infants were pre-term. Human milk and infant microbiota composition are different in mother-infant pairs born pre-term. Milk microbiota has also been shown to differ by lactation stage. Therefore, it doesn’t seem appropriate to compare milk microbiota of mothers who have infants that are 3 and 100 days old. How do you justify the vast differences in both gestational age as well as age in days of infants?

Line 153-154: Did you pool the 4 longitudinal milk samples?

Line 172: What do you mean by overall microbial variation? This contradicts the next sentence, which says that exclusive breastfeeding did not impact diversity or taxa abundance. Furthermore, was there a difference in age of mixed-feeding and exclusive breastfeeding infants? There is a chance that length of breastfeeding, but not proportion of breastfeeding, is influencing composition (i.e. the infant had been suckling for longer).

Line 175-176: Was Bifidobacterium found in these samples from other pairs?

Line 187: The bacteria are not “secreted” into the milk. Please change this wording. Bifidobacterium also likely makes up such a large part of the infant microbiome due to competitive advantage.

Line 196-198: What data are you presenting that support that the milk and infant gut microbiota are seeded through multiple pathways? SourceTracker may have been a good way to demonstrate this but no analysis is included.

Line 200: Please change the words “milk compartment”. Human milk is what was analyzed.

Line 202-204: The difference in abundance of B. breve between the sample types could simply be due to the difference in bacterial load in stool and human milk. Can you elaborate on the statement that B. breve is “selected for” – what exactly do you mean by that?

Line 212: Again, please change wording. Bacteria cannot be secreted.

Figure 1 B: why are other body sites represented in this figure besides maternal and infant stool and breast milk if they are not discussed at all in the results section?

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17 Oct 2019

Reviewer #1: The manuscript describes differences in human milk microbiota composition of mothers whose infants latched and those that had never latched. Data are also presented which suggest vertical transmission of a particular species of Bifidobacterium from the maternal gut to the infant gut by way of human milk. While some of the data are interesting, they seem incomplete. The differences in terms of gestational age and age when samples were collected across infants included in the study are also concerning.

Line 63-64: need citations

We have added the following citations:

Oikonomou G, Machado VS, Santisteban C, Schukken YH, Bicalho RC. Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA. PLoS One. 2012;7(10):e47671.

Contreras GA, Rodriguez JM. Mastitis: comparative etiology and epidemiology. Journal of mammary gland biology and neoplasia. 2011;16(4):339-56.

Gaboriau-Routhiau V, Rakotobe S, Lecuyer E, Mulder I, Lan A, Bridonneau C, et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity. 2009;31(4):677-89.

Ivanov, II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009;139(3):485-98.

Line 63-65: Line is confusing. The existence of an enteromammary pathway is independent ofseeding the infant gut. The enteromammary pathway is from the maternal gut to the mammary gland. It sounds as though you mean that vertical transmission is difficult to prove

This section has been rewritten for clarity, please see revised draft.

Line 75: check the formatting of you bacterial names throughout the paper. This should either be bifidobacteria or Bifidobacterium breve.

Where appropriate, we specified “genus Bifidobacterium”; species “Bifidobacterium breve”; plural Bifidobacteria.

Lines 85-87: The reference provided doesn’t offer much about information about collection processes, and there isn’t enough provided in the manuscript. Was the breast cleaned in any way before collection? How many swabs were used for each sample, and what type of swabs were used? Did mothers wear sterile gloves? Were the swabs that were not frozen immediately refrigerated?

Key elements of the collection process as described below have now been summarized in the methods of the manuscript.

There was no study protocol request for specific cleaning before collection. However, the mother would perform her typical cleaning as if she were to pump for her baby. The rationale behind this was to attempt to capture what the infant was actually exposed to.

One swab per areola for a total of two swabs per patient were collected before the baby latched. The areolar swab kit contained: two (2) snap-tip sterile swabs and two (2) prefilled tubes of transport buffer.

Areolar swab samples were usually taken by the study coordinator who wore standard laboratory gloves, but not surgical sterile gloves. The areolar skin was swabbed in a circular motion starting from the nipple spiraling outwards, avoiding the nipple and the breast skin, for approximately 10 seconds.

Swabs were kept on ice during transport to the laboratory and frozen immediately.

Line 126: No SourceTracker results are presented in the manuscript. This does not need to be included in the methods if no results are presented. If you do choose to include it, what type of data did you put into the program – an OTU table orrarefied sequences?

In the Results section, we refer to SourceTracker results in the text and have now also included a Table 2 with the SourceTracker findings for additional details. We have updated the methods to clarify that the amplicon sequence variant (ASV) table was used as input to SourceTracker.

Line 144: How close to sample collection did the infants receive antibiotics?

This has been clarified in the re-worked section to include details from the clinical data that was collected. Where applicable, we have included maternal intrapartum antibiotics given during delivery and infant postpartum antibiotics in the latched and never latched cohorts as well as when samples were collected. Unfortunately, the duration of antibiotic treatment given to infants post-delivery was not specified.

Table 1: Multiple of these infants were pre-term. Human milk and infant microbiota composition are different in mother-infant pairs born pre-term. Milk microbiota has also been shown to differ by lactation stage. Therefore, it doesn’t seem appropriate to compare milk microbiota of mothers who have infants that are 3 and 100 days old. How do you justify the vast differences in both gestational age as well as age in days of infants?

We agree with the comments that there are differences in milk microbiota based on gestational age and age post-gestation. For the purposes of our analyses, we wanted to pursue the specific question of potential sources of milk microbes between latched vs never-latched infants, so we grouped the totality of evidence from each of these cohorts based on their breastfeeding status. Please note in Table 1 that the latched and never-latched infants ranged from minimum 33 and 34 to maximum 41 and 41 weeks with a median of 39 and 38 weeks gestation, respectively. Indeed, with a larger cohort, stratification into the gestational and post-gestational age-specific demographics would be valuable. Unfortunately, with the small sample size, we could not further stratify meaningfully. Additionally, we show the age of the infant in days in Figure 1 so that the data and its limitations are readily apparent to all readers. We have added this concern as a limitation of our study in lines 249-250.

Line 153-154: Did you pool the 4 longitudinal milk samples?

The four longitudinal milk samples were not pooled. This is clarified in the text: “Of the included never-latched pairs, 1 subject had 4 different milk samples longitudinally collected within the first two weeks of life which were analyzed individually.”

Line 172: What do you mean by overall microbial variation? This contradicts the next sentence, which says that exclusive breastfeeding did not impact diversity or taxa abundance. Furthermore, was there a difference in age of mixed-feeding and exclusive breastfeeding infants? There is a chance that length of breastfeeding, but not proportion ofbreastfeeding, is influencing composition (i.e. the infant had been suckling for longer).

Overall microbial variation was assessed by PERMANOVA, which measures the fraction of variance that can be explained by each covariate. We have clarified this in the methods and results sections. It is possible for a covariate (e.g. exclusive breastfeeding) to explain a significant proportion of variance in microbiome composition but not be associated with significant differences in either diversity or relative abundances.

Duration of breastfeeding may indeed influence the breast milk composition, but we are not able to tease out the role of duration from proportion of breastfeeding given the way the study was designed. Studies that do support breastfeeding exclusivity and percentage influencing the infant gut microbiome (Pannaraj et al, 2017; Ho et al , 2018) have not separated duration from proportion to the best of our knowledge. This would be interesting to investigate in a future, carefully designed study.

Line 175-176: Was Bifidobacterium found in these samples from other pairs?

Only a single mother and her infant had Bifidobacterium (genus)/ Bifidobacterium breve (species) identified in various samples.

Line 187: The bacteria are not “secreted” into the milk. Please change this wording. Bifidobacterium also likely makes up such a large part of the infant microbiome due to competitive advantage.

We have changed “secreted” to “cultivated” or "enriched".

Line 196-198: What data are you presenting that support that the milk and infant gut microbiota are seeded through multiple pathways? SourceTracker may have been a good way to demonstrate this but no analysis is included.

We have now included a Table 2 with the SourceTracker results.

Line 200: Please change the words “milk compartment”. Human milk is what was analyzed.

We have made the suggested change.

Line 202-204: The difference in abundance of B. breve between the sample types could simply be due to the difference in bacterial load in stool and human milk. Can you elaborate on the statement that B. breve is “selected for”– what exactly do you mean by that?

We have simplified the text given that many biological factors could account for the differences and we did not specifically explore the causes for this in our study: “Furthermore, even though Bifidobacterium breve constituted less than 1% of the maternal rectal sample, it made up 28% of the maternal milk sample.”

Line 212: Again, please change wording. Bacteria cannot be secreted.

We have changed “secreted” to “cultivated” or "enriched".

Figure 1 B: why are other body sites represented in this figure besides maternal and infant stool and breast milk if they are not discussed at all in the results section?

The SourceTracker analysis was performed using data from the sample sites included in this figure. We have clarified this in the results section by adding Table 2 describing the SourceTracker results.

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12 Nov 2019

Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve

PONE-D-19-16931R1

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20 Nov 2019

PONE-D-19-16931R1

Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve

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