Human Milk and Infant Intestinal Mucosal Glycans Guide Succession of the Neonatal Intestinal Microbiota
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Review nature publishing group Review Maternal inputs to infant colonization Newburg and Morelli Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota David S. Newburg1 and Lorenzo Morelli2 Infants begin acquiring intestinal microbiota at parturition. which reflects the genotype of the glycosyltransferases of the Initial colonization by pioneer bacteria is followed by active infant, and on the oligosaccharides and glycans of the milk, succession toward a dynamic ecosystem. Keystone microbes which reflects the genotype of the maternal glycosyltransfer- engage in reciprocal transkingdom communication with the ases. The focus of this review is the maternal influences on the host, which is essential for human homeostasis and health; ontogeny of the gut microbiota, that is, how specific human therefore, these bacteria should be considered mutualists milk components affect succession in the bacterial ecosystem rather than commensals. This review discusses the maternal of the infant alimentary canal. Accordingly, we will discuss role in providing infants with functional and stable microbiota. the human milk microbiota, the human milk oligosaccharides The initial fecal inoculum of microbiota results from the prox- (HMOS), and their interaction with the infant gut mucosa and imity of the birth canal and anus; the biological significance of microbiota (Figure 1). this anatomic proximity could underlie observed differences in microbiota between vaginal and cesarean birth. Secondary DEFINING HUMAN MILK MICROBIOTA sources of inocula include mouths and skin of kin, animals The presence of bacteria in breast milk was noted in the and objects, and the human milk microbiome, but guiding 1950s (1,2), with a focus on pathogenic microorganisms. microbial succession may be a primary role of human milk. The Enterobacteriaceae, enterococci, and staphylococci (2–4) were unique glycans of human milk cannot be digested by the infant, the most frequently identified using the plate count technology but are utilized by mutualist bacteria. These prebiotic glycans of the time. Interest in human milk bacteria blossomed in the support expansion of mutualist microbiota, which manifests as 1970s when the use of raw breast milk, especially within neona- differences in microbiota among breastfed and artificially fed tal units, raised concerns about the possibility of milk transmit- infants. Human milk glycans vary by maternal genotype. Milks ting pathogenic bacteria (5,6). Scrupulous procedures for milk Copyright © 2015 International Pediatric Research Foundation, Inc. of genetically distinct mothers and variations in infant mucosal collection and storage are important to achieve “low counts” glycan expression support discrete microbiota. Early coloniza- (below 106 colony-forming units/ml (4,7)). Staphylococcus tion may permanently influence microbiota composition and Pediatr Res aureus, a typical skin inhabitant, was found to be the predomi- function, with ramifications for health. nant organism in freshly expressed milk, while poor storage of the milk was responsible for high counts of enterobacte- 2014 ONTOGENY OF THE GUT MICROBIOTA: OVERVIEW OF ria and other Gram-negative rods. The distribution of bac- SUCCESSION teria was reported for 688 human milk samples (8). In milks 115 Appreciation for the roles of gut microbiota in human health with a quantitative count of <2,500 organisms/ml, the aerobic is rapidly escalating. This is mostly attributable to metage- skin bacteria such as micrococci, staphylococci, and strepto- 120 nomic technologies that allow measurement of the entire cocci (believed safe for consumption) represented 67% of the microbiome, including microbes that are currently not cul- sample. Milks with either a total count >2,500 colony forming 4June2014 turable. Much of the variation in microbiota among individu- units/ml or with a potential pathogen, such as Staphylococcus als is thought to emanate from particular differences in early aureus, β-hemolytic streptococci, Pseudomonas spp., Proteus 4August2014 colonization that have long-term sequelae. At birth, the gut of spp., or Enterococcus faecalis, were deemed to require pasteuri- most infants is largely devoid of bacteria. The inoculation of zation prior to use by infants. This distribution of bacteria did 10.1038/pr.2014.178 the gut and its succession into the adult microbiota is a com- not differ significantly between milks collected at home or in plex process strongly influenced by maternal factors, and the hospital environments. A subsequent bacterial assessment of initial inoculum most closely resembles the maternal fecal milk from 810 individual mothers and 303 pools (9) found that Pediatric Research microbiota. Continued exposure to microbes occurs through- 22% of individual samples did not contain detectable bacteria. out early development. The selection of microbes is dependent However, pools contain the sum of all microbes from each indi- 77 upon the glycan phenotype expressed at the mucosal surface, vidual sample, and 87% of pools contained Staphylococcus; the 1 1Department of Biology, Boston College, Chestnut Hill, Massachusetts; 2Facoltà di Scienze Agrarie, Alimentari e Ambientali, Università Cattolica del Sacro Cuore, Piacenza-Cremona, Italy. Correspondence: David S. Newburg ([email protected]) 12November2014 Received 4 June 2014; accepted 4 August 2014; advance online publication 12 November 2014. doi:10.1038/pr.2014.178 Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 1 | January 2015 Pediatric Research 115 Newburg and Morelli Review Figure 1. Human milk glycans and microbiota influence the ontogeny of neonatal mucosal mutualism. Exposure to maternal and environmental microorganisms at birth coincides with ingestion of maternal glycans in colostrum, especially human milk oligosaccharides (HMOS), which have prebiotic, antiadhesive, and anti-inflammatory activities. HMOS facilitate the selective expansion of mutualist microorganisms, particularly bacteria from the genera Bacteroides and Bifidobacterium, and inhibit growth and adhesion of opportunistic and obligate pathogens at the gut mucosal surface. The expanding mutualistic microbiota stimulate the induction of mucin expression and glycosyltransferase activity (e.g., fut2). Mucosal glycans support a robust, stable microbiota, which benefits the mature host through conversion of dietary components into critical metabolites (e.g., short chain fatty acids) and competitive exclusion of enteric pathogens and occupation of mucosal niches. 2′-FL is 2′-fucosyllactose; LNFP II is lacto-N-fucosylpentaose II; and 6′-SL is 6′-sialyllactose. Gram-positive organisms detected were Enterococcus (16%), introduce other forms of bias; however, for the most part, mod- α-Streptococcus (8%), and S. aureus (4%). In addition, 60% of ern techniques produce results consistent with earlier conclu- pools contained Gram-negative bacteria. Pasteurization kills sions. Denaturing gradient gel electrophoresis analysis after most bacteria, resulting in 93% of pooled milk samples being polymerase chain reaction, when used in conjunction with negative. The presence of skin microbes in cultures of human libraries of 16S rRNA gene sequences, found that the compo- milk led the authors to conclude that the skin microbiota of sition of the microbiome of milk from four mothers (14) was the mother provides the inoculum to breast milk. In a study consistent with the ability of breast milk to provide an inoculum on late-onset neonatal group B Streptococcus infection, the of bacteria to the infant gut. DNA was identified from all skin molecular typing of the group B streptococcus in the infant associated species that had been previously identified in human was identical to that found in the milk samples taken after milk by plating, including staphylococci and streptococci. DNA the infection was detected, suggesting that milk can transmit from lactobacilli and gut microbiota were also detected, but not pathogens to infants (10), or alternatively, that infected infants DNA from Lactobacillus gasseri and bifidobacteria, bacteria can transmit bacteria into their mother’s breast. whose presence had been reported previously from the same These reports conclude that human milk can carry a sub- group when using classical plating techniques. Later, quantita- stantial inoculum of skin-related bacteria, including some tive polymerase chain reaction using specific primers detected pathogens. It is considered prudent to reduce microbes in DNA of streptococci, staphylococci, lactic acid bacteria, and milk with disinfection of the skin and freeze-drying, or, most bifidobacteria in milk collected from 50 mothers (15). Overall, effectively, through pasteurization followed by microbiologic the data from these various techniques confirm the presence testing (11–13). However, pasteurization compromises other and composition of a human milk microbiome, and are consis- aspects of human milk, including the purported presence of tent with human milk providing bacteria to the infant. microbes of potential benefit to the infant that have been more The bacterial composition of milk was assessed at three dif- recently detected through DNA-based culture independent ferent time points from 18 mothers: colostrum obtained within techniques. 2 d, at 1 mo, and 6 mo after mothers gave birth. Milk microbe DNA-based techniques suggest a diverse human milk micro- composition varied by mode of delivery as well as