HMO composition in the milk changes over time and is greatly variable in women delivering prematurely (35). Supplemental Digital Content 4, Table 2, http://links.lww.com/MPG/A964, summarizes changes in class of HMOs and individual HMOs over time in milk, feces, and urine. For most classes of HMOs, the changes over time in the milk were paralleled by similar changes over time in the urine and feces. There were 2 notable exceptions. First, the class of HMOs containing both fucose and sialic acid decreased as a percentage of total HMOs in milk over time, but increased in both absolute numbers and percentages over time in the feces (suggesting minimal consumption by the gut microbes) and increased in absolute numbers in the urine (suggesting absorption from the gut). Second, the class of undecorated HMOs increased in milk over time both in absolute quantity and in percentage of total HMOs, but decreased in percentage in the feces suggesting that these structures were consumed by the gut microbes and/or absorbed from the gut (consistent with the increase in absolute numbers in the urine). Several individual HMO structures appeared to be consumed in the gut (present in milk but decreased over time or absent in the feces, eg, DFLNO I and 6'Sialyllactose [SL]) or readily absorbed from the gut and filtered into the urine (present in milk and in urine, eg, lacto-N-tetraose + lacto-N-neotetraose and MFLNH I + III).
Simple linear regression analysis comparing the fecal microbial classes other than bifidobacteria to the fecal composition of HMO classes yielded few significant associations. Among the second stool samples, for Enterobacteriaceae, no correlations were noted, whereas for Bacteroides, there was a weak positive correlation with percentage fecal undecorated HMOs (Supplemental Digital Content 6, Fig. 3, http://links.lww.com/MPG/A966).
In term infants who receive predominantly their mother's own milk, bifidobacteria are common colonizers of the infant gut accounting for up to 90% of total intestinal bacteria in developing countries (36). In western countries, the percentages of bifidobacteria are lower, even in healthy breast-fed infants who were born vaginally and have not been exposed to antibiotics (37,38). These differences likely play a role in the many compelling observations supporting the hygiene or “old friends” hypothesis, including the increased incidences of type 1 diabetes and food allergies seen in developed countries (38). In premature infants, bifidobacteria are conspicuously absent even when the diet is exclusively human milk (33). Differing species of bifidobacteria vary in their capacity to colonize the premature infant, based in large part on their capacity to use HMOs as a nutrient source (22). Differences in bifidobacteria at the subspecies and strain level have been characterized (39,40). Probiotics have been shown in several cohort studies and placebo-controlled randomized clinical trials to decrease the risk of NEC with several Bifidobacterium species demonstrating efficacy (41). We have previously demonstrated colonization and a decreased incidence of NEC in a cohort study of the B breve strain administered to this cohort (42,43). The largest clinical trial to date of probiotics for the prevention of NEC in premature infants found no difference between B breve and placebo (44). In that trial, the administered strain differed from the one studied herein, the dose administered was low compared to other similar trials, cross-contamination between the treatment and placebo groups was common, and the infants who were colonized with the probiotic (as opposed to the infants that received the probiotic) had lower rates of NEC, late-onset sepsis, and death (45).
The dominance of Enterobacteriaceae in this cohort is consistent with other reports (33) and is important given the compelling evidence that members of this family of bacteria include Klebsiella pneumonia and Escherichia coli, both of which have been demonstrated to predispose to NEC. In a previous probiotic trial in premature infants, we found a decrease in fecal Enterobacteriaceae with administration of B longum subsp infantis but not with B animalis subsp lactis (22). In this cohort, the percentage of Enterobacteriaceae increased over time in spite of B breve administration.
In a previous study of a cohort of premature infants in the United States, we demonstrated that the composition of HMOs in the mother's milk influences the fecal microbiota and that there are significant differences among HMO structures in absorption from the gut, excretion in the urine, and consumption by gut microbes. In that study, the infants had not received any probiotic or prebiotic supplements. There were correlations between the class of fucoslyated HMOs in the milk and decreased Enterobacteriales in the infant feces (significant individual HMO structures included 2’FL, lactodifucotetraose, and lacto-N-fucopentaose V) and between the class of undecorated HMOs in the milk and increased Enterobacteriales in the infant feces (1 significant HMO structure: lacto-N-neohexaose). As expected, the primary consumers of HMOs (bifidobacteria and Bacteroides) were present in very small amounts (46). The current study differs in that all of the infants received the probiotic B breve with most of the infants showing colonization by the time of the second sample. Regardless of how we defined “responders” and “nonresponders,” we found similar associations with undecorated HMOs more abundant in the milk fed to responder babies and total fucosylated HMOs more abundant in the milk fed to non-responders. The most likely explanation is that the undecorated HMOs are indeed a significant part of the explanation as to why some premature infants become colonized by probiotic B breve, whereas others do not. Likely HMO candidates include lacto-N-hexaose, lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-neohexaose (see Supplemental Digital Content 3, Table 1, http://links.lww.com/MPG/A963 and Supplemental Digital Content 5, Table 3, http://links.lww.com/MPG/A965). It is also likely that some structures that contain both fucose and sialic acid and some structures with fucose but not sialic acid are not able to be consumed by the bifidobacteria present in these babies (including the probiotic B breve). A high abundance of these structures in the milk would mean fewer HMOs that are available as a food source for the bifidobacteria resulting in poor colonization with the probiotic. This is consistent with the data in Table 3 wherein the classes of fucosylated, fucosylated + sialylated, and total fucosylated and the single HMO MFpLNH IV were all associated with an increase in the log feces:milk ratio over time. In our previous study, we saw trends toward differences in the infant fecal microbiota based on maternal secretor status, but this was not seen in the current larger cohort.
Over 100 HMO structures have been identified (75 of these structures are presented in Table 3 of a recent methods paper (29)). We selected 46 of the most abundant structures for the present study. Several HMO structures are worthy of individual comment. First, 2’FL, which is abundant in the milk of secretor mothers but essentially absent from nonsecretor mothers, can be synthesized at a reasonable cost, and is now added to some infant formulas. This HMO is consumed in vitro by only 5 of 26 strains of B breve (14). A recent study in the premature piglet model found no alteration in the intestinal microbiota, digestive function, or incidence/severity of NEC with addition of 2’FL to the diet (47), whereas in the rodent model, 2’FL is protective against NEC and increases mesenteric perfusion (48,49). In the current cohort, we found no changes in 2’FL in the milk, urine, or feces over time, no significant differences between fecal bifidobacteria based on maternal secretor status, and no associations between maternal milk 2’FL content and the intestinal microbiota, all suggesting that the administered strain of B breve is not a consumer of 2’FL. Second, 3FL is found in the milk of both secretors and nonsecretors, was consumed by 10 of 26 strains of B breve (14), increased in the milk over time, but was not found in significant quantities in many of the stool specimens (suggesting consumption by gut microbes) or urine specimens (suggesting a lack of absorption from the gut). This was confirmed by the almost 20-fold decrease in the log fecal:milk and urine:milk ratios over time (Table 3 and Supplemental Digital Content 5, Table 3, http://links.lww.com/MPG/A965), suggesting that this HMO is consumed by the administered B breve strain. Third, DSLNT (disialyllacto-N-tetraose) has been demonstrated to decrease NEC incidence/severity in the rodent model (50). In the current cohort and in our previous cohort, DSLNT was in very low abundance in all specimens. Fourth, the abundant undecorated HMO LNT is aggressively consumed by all 26 tested B breve strains (14), increased in the milk over time, decreased in the feces over time (suggesting consumption by gut microbes) and increased in the urine over time (suggesting absorption). Finally, B breve strains in general are able to utilize large but not small sialylated HMOs (14). In the present study, the large HMOs, sialyllacto-N-tetraose isomers b and c decrease over time in the milk, are uncommon in feces (suggesting consumption), and increase over time in the urine (suggesting absorption), whereas the small HMO 3'SL decreases over time in the milk, but does not change over time in the feces or urine (suggesting a lack of both consumption and absorption). Interestingly, 6'SL, also a small sialylated HMO, appears to be consumed by the gut microbes in this cohort.
The authors express appreciation to Dr. Abe and Morinaga Milk Industry Co, Tokyo, for provision of the probiotic B breve.
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