What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study

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K.A. Lackey, J.E. Williams, C.L. Meehan, J.A. Zachek, E.D. Benda, W.J. Price, J.A. Foster, D.W. Sellen, E.W. Kamau-Mbuthia, E.W. Kamundia et al.

COMMENT: This work reports important results from the INSPIRE Study about the 16S rRNA taxonomic profiling analysis of almost 400 samples of mother (milk) and child (feces) matched-dyads collected from different countries and environments: rural Ethiopia, urban Ethiopia, rural Gambia, urban Gambia, Ghana, Kenya, Spain, Sweden, Peru, California (United States) and Washington (United States). “Core” and “unique” cohort-specific bacteria are described for mothers milk microbiomes and their infant fecal microbiomes.


To describe microbiomes of milk produced by relatively healthy women living at diverse international sites and compare these to the fecal microbiomes of their relatively healthy infants


These are some of the ample set of interesting results of this work:

A set of “core” genera were characterized for each sample type both in the overall dataset and within each cohort. To be included in the core taxa, a genus must have been present in ≥ 90% of the samples and represent ≥ 0.1% of all identified taxa. We also characterized the relatively “unique” taxa across all taxa identified in the sequencing run for each sample type, defined as genera present in only one cohort and in ≥ 10% of samples within that cohort.

Infant Fecal “Core” and “Unique” Bacteria

Overall, Streptococcus, Escherichia/Shigella, and Veillonella were identified as core taxa in infant feces, being present in 98.4, 91.7, and 90.2% (respectively) of all samples . When considered within each cohort, there were sometimes different sets of core taxa. For example, Lactobacillus was part of the core taxa for ETR, GBR, GN, and KE, and Bacteroideswas part of the core taxa for USW. There were no unique bacterial genera identified within any cohort.

Milk Microbiome: Core and Unique Bacteria

For milk, Staphylococcus and Streptococcus were identified as core genera, being present in 98.7 and 97.7% of all samples, respectively (Table 3Supplementary Figure 13). However, as with feces, there were sometimes different sets of core taxa within each cohort: Propionibacterium was also present in 90.5% of milk from KE, Dyella in 94.9% of milk collected in USW, and Corynebacterium 1 in 95.0 and 94.1% of milk samples collected in ETR and ETU, respectively. In addition, it is noteworthy that milk collected in ETR contained 8 core genera, including Rhizobium, Brevundimonas, and Achromobacter, which were present in every sample from this location. There were also several unique bacteria identified in some cohorts in milk: only milk collected in ETR contained Acidothermus, Demequina, Flaviflexus, and Pediococcus; milk from GBU uniquely contained Chroococcidiopsis and Isoptericola; and milk from GN uniquely contained Akkermansiaand Butyricicoccus.

Microbial Relationships Between Women and Their Infants

Upon evaluating the relationship between a mother's milk microbiome and her infant's fecal microbiome using both the Jaccard and Bray-Curtis distance metrics, the mother/infant dyads' samples were found to be more similar or tended to be more similar to each other than to all other combinations of mothers and infants (with Jaccard index: P = 0.0258; with Bray-Curtis dissimilarity: P = 0.0936). When evaluated within each cohort, microbial communities of a mother's milk and her infant's feces were not more similar to each other than to all other combinations of mother/infant dyads (all P > 0.05 for both matrices), except for in mother/infant samples from ETU in which the Bray-Curtis distance metric showed that bacterial communities between mothers and their infants were more similar to each other (P = 0.0185) than to other random combinations within the cohort.


The amplicon region of the bacterial 16S rRNA gene was sequenced  was the V1-V3 region using Illumina MiSeq paired-end 300-bp protocol for 600 cycles.

Only R1 read was used for the analysis:

Reads with < 270 bases were discarded. Because of loss of reads and little overlap between forward and reverse reads following quality filtering and trimming, only forward reads were used in subsequent analyses.

After additional filtering of any read that could not be classified to the genus level, and omitting any sample with < 1,000 reads, the infant fecal dataset analyzed here contained 4,382,947 reads across 377 samples, with a mean (± SD) of 11,626 ± 6,274; the range did not change. For the 409 milk samples, sequencing generated 7,529,453 reads, with a mean (± SD) of 18,967 ± 18,646 reads and a range of 1,391–141,620 reads. Using the same filtering criteria as the infant fecal dataset, the milk dataset used here contained 6,709,277 reads across 394 samples, with mean (± SD) of 17,029 ± 16,783, and a range of 1,302–130,700 reads. These curated datasets were used for all further analyses.

Perhaps, a more specific taxonomic assignments would have been possible using the sequence data from both R1 and R2 and, at the same time, it could have avoided the possible bias caused by discarding all the reads that could not be classified to the genus level.

The authors commented about it at the discussion:

In this case, we also were only able to use the forward read, which could have been a source of bias in elucidating the microbial genera present.

Probably new analysis of these sequencing data will provide new enriched results for this interesting and important study.


In conclusion, this study is the largest of its kind to use standardized methodologies to characterize and compare the milk and infant fecal microbiomes of maternal/infant dyads worldwide. Substantial differences in both the composition and relative abundance of specific taxa were present among cohorts. We also found substantial variation among women/infants within a cohort, suggesting that environment alone does not drive variation in milk and fecal microbial community structure.

… we conclude that what is “normal” in terms of the fecal microbiome of healthy infants and milk microbiome of healthy lactating women varies by culture and/or location. Further, we posit that bacterial compositions of human milk and feces of breastfed infants are likely specific to a dyad within a culture and location


ETR: rural Ethiopia. ETU: urban Ethiopia. GBR: rural Gambia. GBU: urban Gambia. GN: Ghana. KE: Kenya. SP: Spain. SW: Sweden. PE: Peru. USC: California (United States). USW: Washington (United States).


Raquel Tobes