The results from this study suggest that HMOs consumed by infants and the associated microbiota both impact infant health outcomes. These associations with proxy health measures in the present study comprise context-specific correlations, which at present have only theoretical causational support. However, this study is valuable as an initial hypothesis-generating screen from which to direct future research efforts into mechanisms. The data indicate that mothers of infants who had zero sick days (reported instance(s) of diarrhea, fever, rash, coughing, etc.) produced milk with lower relative amounts of LNT and higher relative amounts of LNFP I and III compared to mothers of infants who had sick days. Isomers LNFP I and III could not be chromatographically separated during analysis, but since LNFP III is <5% the abundance of LNFP I, discussion was focused on LNFP I5. LNFP I is the fucosylated version of LNT, with Fuc in an α(1-2) linkage on the terminal Gal. F-LNO at week 4 and MFpLNH IV and IFLNH III at week 16 were also different between sick and not sick infants, but these fucosylated structures were found at higher relative abundance in the sick infants. The Fuc linkage on all three of these structures is α(1-3), unlike LNFP I which is an α(1-2) linkage, and they are found at much lower abundance (less than 5%) than LNFP I which makes up around 12% of HMOs. Total relative fucosylation was not significantly different between sick and not sick infants. Previous studies have also shown that higher consumption of α(1-2) linked Fuc led to lower experiences of diarrhea26. Newburg et al. showed a decreased incidence of diarrhea in infants who consumed milk with higher ratios of α(1-2) linked Fuc compared to HMOs with α(1-3/4) linked Fuc26. Previous studies have found 2′-fucosyllactose (2′FL, Fuc(α1-2)Gal(ß1-4)Glc) to be associated with reduced diarrhea incidence caused by Campylobacter jejuni, an infant gastrointestinal pathogen36,37,38. In this study we did not observe any statistically significant associations between reported morbidity and 2′FL. Instead, our results showed that it was the α(1-2) fucosylation of LNT to LNFP I that was associated with lower morbidity. Together, these findings point to specific roles for different α(1-2) fucosylated structures in the protection from morbidity.

Statistical models were used to predict WAZ and HAZ scores at 20 weeks as a response to the HMO abundances. Relative 3′SL (Neu5Ac(α2-3)Gal(ß1-4)Glc) was the strongest model predictor, contributing positively to WAZ, meaning the higher the proportion of 3′SL produced by the mother from 4 to 20 weeks, the larger her infant’s WAZ score at 20 weeks was. LSTc (Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc), on the other hand, contributed negatively to WAZ at 20 weeks. Relative total sialylation was not a predictor. As with the positive associations between α(1-2) but not α(1-3) fucosylated structures and morbidity, again here, an α(2-3) but not an α(2-6) sialylated species was associated with better growth. Infants from a Malawian cohort who were severely stunted at 6 months postpartum had mothers who produced significantly less sialylated HMO than mothers with infants of healthy height39, agreeing with our findings here of a positive association between sialylated HMO and growth. Increased weight gain was also observed in model mice and piglets fed diets with supplemented sialylated bovine milk oligosaccharides, mainly 3′SL and 6′SL (Neu5Ac(α2-6)Gal(ß1-4)Glc)39.

In addition to 3′SL, LNFP I was also found to contribute positively to growth, in this case HAZ, at 20 weeks. As indicated earlier, LNFP I was found at higher abundance in the milk of infants who did not have sick days. These findings suggest that LNFP I may help infants maintain growth by sparing resources that would otherwise be redirected toward fighting off infection. Whether these changes in relative concentration of LNFP I are indicative of upregulation of the FUT2 gene, its activity, and/or other compositional changes in milk that are then responsible for protecting the infant from infection, or whether LNFP I directly protects the infant by binding and inactivating pathogens in the intestine, needs to be determined in future studies.

The mothers’ HMO profiles were significantly affected by the seasonal changes experienced in The Gambia. Prentice et al. showed that during the dry season, lactating mothers had an increased energy intake compared to mothers lactating in the wet season, which could explain the ability of mothers to produce milk with higher concentrations of HMOs when nursing in the dry season40. Previous research has also shown seasonal variation in overall milk output, not just HMO output, with a reduced amount of milk consumption during the wet season, concomitant with a higher risk for infection and growth failure in infants born during the wet season41,42. HMO production decreases throughout lactation, but there could be a potentially larger decrease in HMO output by mothers nursing in the wet season at 20 weeks postpartum due to their reduced caloric intake. This could be detrimental to their infant’s growth and morbidity status. It is important to note, however, that as the infant grows older he/she consumes a higher total volume of milk per day and therefore the total dose of oligosaccharides per day may remain consistent, although they are consuming a less concentrated milk. Total volume of milk consumed by each infant was not measured in this study. Although the majority of infants were exclusively breast-fed through 20 weeks, the introduction of semisolid foods and water could also affect milk intake and milk composition. The mechanisms by which a seasonal energy deficit in the mother is translated to HMO production levels in the mammary gland are currently unknown and deserve further study to better understand how infant needs are communicated to the mother to alter milk composition to meet those needs.

In this study we chose to analyze the HMO data as relative rather than absolute abundances of HMO, because as noted above, the total amount of milk consumed by each infant each day was not measured, therefore the total abundance of HMO consumed by each infant could not be calculated. However, we also focused on relative abundances because we were interested in how changes in the composition of HMOs affect health outcomes. The compositional differences are indicative of fluxes through the glycosyltransferase pathways. For example, an increased relative concentration of the fucosylated structure LNFP I with a concomitant decrease in the relative concentration of its precursor LNT is indicative of an increased flux in α(1-2) fucosylation. The increased α(1-2) fucosylation of specific HMO in this cohort was associated both with protection from morbidity and with improved growth. Mechanistic studies are needed to understand how seasonal, dietary, environmental, genetic, and/or other factors influence the ability of the mammary gland to turn on α(1-2) fucosylation in an individual mother to increase protection of the infant from infection. Similar mechanisms may be involved in the regulation of the production of sialylated structures and growth.

As observed in other cohorts of infants from less-developed countries like Venezuela, Bangladesh, and Malawi, bifidobacteria (and specifically B. infantis) were the dominant microbe in this group of infants10,43,44. B. infantis possesses a large genetic complement of enzymatic tools to consume the major human milk glycans in breast milk, suggesting that the dominance of the gut community by this microbe is driven by the availability of these substrates17,19,20. Indeed, in this study, we found that B. infantis was the only (sub)species positively correlated with LNnT, which represented ~10.1% of total HMO. The observed shifts in microbial community composition over time (class Bacilli being more abundant at 4 weeks, the Bacteroidetes phylum, Proteobacteria phylum, and Coriobacteriia class more common at 16 weeks, and the Clostridia class more abundant at 20 weeks) may represent the succession of species more acclimated to environmental conditions promoted by weaning from an exclusively breast milk diet, and/or changes in the microbial exposure of the infant as he/she starts to move and sample their environment. Of the 23 infants whose microbiota was measured at all three time points, seven had received water, cow’s milk, and/or semisolid food by week 20. Three infants were also fed from a bottle for some of their meals rather than feeding at the breast, which could also contribute to bacterial diversity at 20 weeks. Initially colonizing Lactobacilli (of possible vaginal origin) present at 4 weeks may be replaced by both Bacteroides species able to consume select HMOs and by species that can use the metabolic end products of the dominant bifidobacteria45,46. A shift toward higher amounts of Clostridia is also associated with the consumption of weaning foods47,48.

Colonization by bifidobacteria has been associated with proxies for desirable infant health outcomes such as higher resistance to pathogen colonization, improved response to some vaccines, better gut barrier function, enhanced immune surveillance, and reduced inflammation9,10,12,13,14,15. However, we found no correlations between bifidobacteria levels and morbidity, likely because of the ubiquity of bifidobacteria in this cohort. We did find, however, that infants who did not have sick days had higher Prevotella, which agrees with a previous study in a cohort of children from Malawi44. Although there were too few such cases in this cohort of 33 infants to allow for statistical analysis, several infants had large shifts in the total amount of bifidobacteria at certain time points, which coincided with changes in growth patterns and/or morbidity measures. For example, infant 25 started out with over 60% bifidobacteria at weeks 4 and 16, which fell to less than 10% bifidobacteria and was largely replaced by Enterobacteriaceae at week 20. Although she had not had any sick days at either week 4 or week 16, at week 20 this infant had 12 sick days, a very high calprotectin level (867.67 mg/kg), and lost weight from week 16 to week 20. Following a similar pattern, the relative abundance of LNFP I in her mother’s milk went from 24% at week 4 to 18% at week 16, to a very low level of 0.1% at week 20. At the same time, the relative abundance of 2′FL stayed about the same (12%, 12%, 11%), whereas the relative abundance of LNT increased from 15% at week 4, to 26% at week 16 and 23% at week 20. The overall composition of milk decreased 12% in fucosylated structures but increased 10% in undecorated HMOs. Percent sialylation varied from 12% at week 4 to 9% at week 16 to 17% at week 20. In this particular case, the infant was born in the dry season, but mother and infant transitioned into the wet season in week 16 and were still in the wet season at week 20. These findings indicate that significant shifts in the mother’s HMO milk composition coincided with both the shifts in the infant’s gut microbiota and with the infant’s growth and morbidity outcomes (see Supplementary Fig. S8). They also highlight the seasonally-driven shifts in resource availability for mothers, and again point to LNFP I as a potentially important indicator of health outcomes.

Our finding that Bacteroides was increased in those infants who had high fecal calprotectin levels is intriguing in light of several recent reports. High levels of Bacteroides colonization are common in many of the cohorts of infants from developed countries studied so far, and are more common in vaginally-delivered infants25,49,50. Whereas B. infantis is specifically specialized to consume a broad array of HMOs17,51,52, Bacteroides species are also able to consume at least some HMOs, though select bifidobacteria outcompete Bacteroides for these carbon sources46. Our results demonstrate that in the background of a dominant bifidobacterial population, increasing amounts of Bacteroides species are associated with increased neutrophil-related gut inflammation as measured by fecal calprotectin levels. Vatanen et al. showed that Bacteroides presence may expose infants to increased amounts of lipopolysaccharide (LPS) types which are linked to downstream autoimmune disease by inhibiting the immune-stimulating effects of E. Coli derived LPS53. It is unknown whether the Bacteroides species present in the Gambian infants studied here possessed LPS types similar to the Bacteroides dorei studied by Vatanen et al. Bacteroides degradation products of sialylated milk oligosaccharides have been shown to promote the growth of inflammation-inducing (and potentially pathogenic) Enterobacteriaceae in in vitro studies, however we did not detect an enrichment of enterobacteria in infants with abnormally high calprotectin levels39,54,55.

Our data show a positive correlation between Lactobacillus and fucosylated structures; an observation supported by a study showing that Lactobacillus casei BL23 contains genes encoding fucosidases which were capable of degrading fucosylated oligosaccharides56. Other lactobacilli, however, such as Lactobacillus delbrueckii and L. rhamnosus show very little growth on 2′FL57. Our findings of positive correlations between Streptococcus and total sialylation, fucosylated and sialylated structures, and LSTc match a report that several strains of Streptococcus have been shown to produce sialidases58. Other streptococci strains, however, are negative for sialidase activity59. B. longum subsp. longum was negatively correlated with fucosylated HMO, confirming previous observations25. B. longum subsp. longum is likely less efficient at consuming fucosylated HMOs due to its lack of HMO-consumption related genes, including fucosidases, thus higher breast milk fucose content would be predicted to lead to lower colonization by B. longum subsp. longum19. B. infantis was the only microbe that was found to be positively correlated with LNnT, which confirms previous reports that B. infantis grows very well on this particular HMO species18. Thus the ability of the microbiome to degrade certain types of oligosaccharides is dependent on the specific strains comprising the infant’s microbiota. This strain variability may contribute to our failure to find functional differences in the microbiomes between infants fed by mothers of different secretor status using the predictive tool PICRUSt, as it is dependent on the specific sequenced strains available in public databases. Other factors must be taken into consideration when discussing the significance of these associations, such as cross-feeding between strains, growth of certain taxa on other microbial by-products, the influence of other breast milk components, and the fact that a known HMO-consuming bifidobacterial strain was dominant in the majority of these infants. Although our sequencing-based results do not test the HMO-consumption-related metabolic abilities and low-level enzymatic functions of the specific microbial species present in these infants, the correlations we present are in many cases consistent with the known abilities of studied strains. However, we cannot rule out the hypotheses that these observations are due to unrelated microbe-microbe interactions or co-correlation with a third factor rather than direct consumption of HMOs by the correlated microbe.

To evaluate the impact of microbial differences between classes of infants, the functional abilities of the fecal microbiome were predicted using PICRUSt. While no significant differences were found that might explain infant growth or health outcomes, three functions were found to increase over time. One of these functions, butyrate-acetoacetate CoA-transferase, can be involved in butyrate metabolism, and in the context of infant gastrointestinal tracts is likely involved in utilizing the larger concentrations of bifidobacterial-derived acetate present in the environment45,60,61,62,63. The largest contributor of this function was the Veillonellaceae. This shift likely reflects the maturation of the microbiome community as selective pressures assert themselves and niches are filled by better-adapted species.

Together these results lead us to conclude that changes in milk HMO composition, as well as shifts in the infant’s gut microbiota are both associated with infant health outcomes in The Gambia. Specifically, the relative abundance of LNFP I, the α(1-2) fucosylated structure of the parent compound LNT, was found to be higher in infants who did not have sick days compared to those who had sick days, and was also predictive of HAZ score at 20 weeks, suggesting that this HMO may be either directly or indirectly involved in protecting the infant from infection and thus promoting growth. The α(2-3) sialylated structure 3′SL was predictive for infant weight-for-age. Our findings suggest that specific HMOs and/or specific glycosyltransferase pathways can alter the composition of milk toward a more protective profile which is associated with lower rates of infection/inflammation, and which allow the infant to invest energy in growth. In this cohort, as with other cohorts in similar countries, infant gastrointestinal tracts were dominated by bifidobacteria. We were not able to detect statistically significant effects of shifts in bifidobacteria abundance because for most infants bifidobacteria levels stayed high (on average about 70% of total bacteria) across all three time points, although in five out of seven cases when bifidobacteria levels dropped significantly, there were concomitant increases in morbidity and/or growth faltering, as well as large shifts in HMO profiles. Prevotella was associated with decreased morbidity. In this study, we found that the seasonal fluctuations in energy availability in The Gambia affected the mother’s ability to produce HMOs such that mothers lactating during the wet (hungry) season had larger decreases in total HMOs over time than mothers lactating during the dry season. How the overall health, environment, and diet of the mother affects the quality of her milk and thus the ability of her milk to protect her infant from infection and promote growth is an important question to resolve in future studies. Mechanisms involved in mediating the protective effects of HMO profiles and specific HMO structures on infant morbidity and growth also need to be further studied, as do the gut microbiota-specific pathways involved in mediating these effects.