breast milk , the sole source of nourishment 
 for newborns , has been 
 under intense selective pressure over millions of years of evolution 
 to meet the infant s needs to grow and survive . interestingly , the third most abundant chemical component class 
 in human milk after lactose and lipids , human milk oligosaccharides 
 ( hmos ) , is both non - nutritive and non - digestible . hmos play a key role in creating and maintaining a healthy 
 infant gut microbiota through two established mechanisms . first , hmos 
 have prebiotic effects ( promoting growth of beneficial bacteria such 
 as bifidobacterium and lactobacillus ) . recent 
 metagenomic studies have confirmed that bifidobacteria are enriched 
 in the intestines of healthy breast fed infants . in vitro studies provide a reason for the prevalence of 
 bifidobacterial and even bacteroides species : they selectively consume 
 hmos . a number of studies have characterized the 
 enzymes in bifidobacteria that specifically degrade hmos ( reviewed 
 in ref ( 8) ) . second , hmos compete for specific pathogen binding with sites in 
 the infant gut . free hmos and conjugated 
 cell surface glycoforms are synthesized by similar glycosyltransferases 
 and thus have common epitopes . ingested hmos can thus interact with 
 enterocyte surface molecules , limiting binding to these sites by viruses 
 such as hiv and rotavirus . in addition , because hmos are also unbound , 
 they can serve as free analogues of pathogen host receptors . instead 
 of binding to cell surface glycoproteins or glycolipids , pathogens 
 bind to hmos . anti - adhesive activity 
 of free hmos has been described for vibrio cholerae , salmonella fyris , enteropathogenic and enterotoxigenicescherichia coli , streptococcus pneumoniae , and caliciviruses . for instance , fucosylated 
 hmos inhibit the binding of campylobactor jejuni to intestinal cells , whereas sialylated 
 hmos block the adhesion of e. coli to 
 human erythrocytes . the functions 
 of hmos are related to their specific structures ; 
 however , identifying and quantifying those structures has been a major 
 challenge in hmo analysis . hmos can have diverse and complicated structures 
 even though they commonly share a lactose core consisting of glucose 
 and galactose linked via a -1,4-linkage . functional studies to date have involved either a limited 
 number of structures or little or no structural information . the benefits of breast feeding include the long - term health implications 
 of a well - established , specific , and protective microbiota . recent 
 advances in glycomic analysis and genomic sequencing have allowed 
 us to observe the correlation between hmo consumption and gut microbiota 
 populations . in this study , we demonstrate two examples of direct 
 correlation between the abundances of hmos in feces and the composition 
 of the infant fecal microbiota as proof - of - concept . hmos from fecal 
 samples of breast - fed infants were analyzed using advanced separation 
 and mass spectrometry methods that allow quantitation of specific 
 structures . microbial populations were characterized by next - generation 
 sequencing of 16s rdna amplicons , specific qpcr , and bifidobacterium species - specific terminal restriction 
 fragment length polymorphism ( bif - trflp ) . the observed shift in the 
 gut microbiota of these two healthy infants from a non - hmo - consuming 
 population into a more saccharolytic microbiota with a corresponding 
 decrease in fecal hmo intensities supports more extensive analyses 
 of larger populations . the study was approved 
 by the institutional review board at uc davis ( protocol no . 200715509 - 4 ) , 
 and informed consent was obtained from the parents of infants prior 
 to participation . the infants included were born vaginally at term 
 and were not treated with antibiotics during the study period . infant b had formula supplementation 
 for 4 days right after birth , from days 2 to 6 , and then was solely 
 breast fed . details of the mothers diet and health status 
 were not collected as part of this study . feces samples were 
 collected from two full - term infants as follows : twice a week for 
 the first month , twice a month in the second month , and once or twice 
 a month thereafter . crude hmos were isolated 
 and purified from extracted feces through 
 a series of liquid- and solid - phase extractions , as described previously . briefly , four volumes of chloroform / methanol ( 2:1 v / v ) were added 
 to the decanted liquid , and the mixture was centrifuged at 4000 g for 30 min at 4 c . two volumes of ethanol were added , and the mixture was 
 left at 4 c overnight and then centrifuged for 30 min at 4 c . the supernatant solution was evaporated to dryness using a centrifugal 
 evaporator ( savant aes 2010 ) . hmos were reduced to alditol form 
 by adding sodium borohydride and incubating at 65 c for 1 h , 
 purified by solid - phase extraction using c8 and graphitized carbon 
 columns , and evaporated to dryness . oligosaccharides were profiled using matrix - assisted laser desorption / ionization 
 fourier transform ion cyclotron resonance mass spectrometry ( maldi 
 ft - icr ms , agilent , formerly varian , palo alto , ca , equipped with 
 an external promaldi source ) and nano - high - performance liquid chip / time - of - flight 
 ( nano - hplc - chip / tof , agilent 6200 ) ms , as described previously . for maldi ft - icr ms experiments , 2,5-dihydroxy - benzoic acid ( dhb ) 
 was used as matrix ( 5 mg/100 l in 50:50 acn / h2o ) 
 and sodium chloride ( 0.01 m in 50:50 acn / h2o ) was used 
 as cation dopant . the precursor ions were detected primarily as [ m 
 + na ] ions in the positive ionization mode . identification 
 of hmo compositions was done based on accurate masses using glycan 
 finder software ( written in - house in igor pro 5.04b , wavemetrics ) , 
 which filters experimental accurate masses to a specified mass tolerance 
 ( set at 20 ppm ) based on accurate masses of theoretical oligosaccharides . the nano - hlpc - chip / tof ms experiments , as described previously , were performed using a porous graphitized carbon hplc microchip 
 with a 40 nl enrichment column and 43  0.075 mm i.d./5 m 
 pore size analytical column . separation was achieved using a binary 
 gradient solvent system with a ( 3% acn in 0.1% formic acid solution ) 
 and b ( 90% acn in 0.1% formic acid solution ) . the chip column was 
 equilibrated and eluted at a flow rate of 0.4 l for nanopump 
 and 4 l for capillary pump . the gradient ran for 65 min and 
 was programmed as follows : 2.520 min , 016% b ; 2030 
 min , 1644% b ; 3035 min , b increased to 100% ; 3545 
 min , continue at 100% b ; and 4565 min , 0% b to allow equilibration 
 of the column prior to the next sample injection . data were acquired 
 in the mass range of m / z 2003000 
 using electrospray ionization ( esi ) in the positive ionization mode . deconvoluted peaks were extracted using the molecular feature of the 
 agilent mass hunter software at 20 ppm error . ms searcher software ( in - house , written in java ) , which assigns structures 
 to a peak based on retention time and accurate mass . lc  ms 
 searcher uses a library of 45 neutral hmos and 30 acidic hmos characterized 
 using exoglycosidases and ms / ms by wu et al . around 2000 spectra are inputted in the program per run , and with 
 the hmo library , the lc ms searcher software identifies hmos 
 from all of the scans based on retention times and m / z values . it then outputs intensities , structures , 
 and deuterium / hydrogen ( d / h ) ratios of said hmos . this library approach 
 allows for separation of isomeric species and identification of individual 
 structures based primarily on accurate masses and reproducible retention 
 times , since extensive confirmation of hmos by ms / ms and exoglycosidases 
 were done previously . deuterium - labeled internal standards were 
 used for the relative quantification of hmos using maldi ft - icr ms , 
 as described previously . this method 
 gives a relative standard deviation of less than 15% and allows for 
 a more linear quantitation over a larger dynamic range . briefly , pooled hmos isolated from milk of several 
 donor mothers were reduced using sodium borodeuteride , purified by 
 graphitized carbon solid - phase extraction , and used as reference solution . both the deuterated reference solution and sample solution were spotted 
 as one sample onto the maldi plate and analyzed by maldi ft - icr ms 
 in the positive mode ( n = 3 ) . the ratio of sample 
 reduced by nabh4 and standard reduced by nabd4 ( h / d ratio ) was calculated using microsoft excel using the formula 
 described below . h / d ratios were used for quantitation as described 
 by ninonuevo et al.where m and n are the experimental intensities of a ( monoisotopic 
 peak ) and a + 1 , respectively , and p and q are calculated intensities of a and a + 1 , respectively . to compute the temporal 
 changes in the h to d ratios across a set 
 of samples , a reference sample is set and is used to compare with 
 all the other samples to quantitate oligosaccharide to the isomer level , both the 
 deuterated 
 reference solution and sample solution were also injected into the 
 nano - hplc - chip / tof ms . ms 
 searcher ( the program outputs d / h ratios ; 
 we took the reciprocal ) and verified manually using microsoft excel . bacterial genomic dna was 
 extracted from fecal samples in order to analyze the infant gut microbial 
 populations as previously described with a few modifications . briefly , 200 mg of stool was resuspended 1:10 
 in ice - cold phosphate buffer solution ( pbs ) and centrifuged ( 8000 g for 5 min at room temperature ) ; then , the supernatant 
 was decanted . this rinse was repeated twice , and the fecal pellet 
 was resuspended in 200 l of lysis buffer ( 2 mm edta , 1.2% tritonx-100 , 
 20 mm tris - hcl , ph 8.0 ) with freshly added 40 mg / ml lysozyme . buffer asl from qiaamp 
 dna stool mini kit ( qiagen , valencia , ca ) was added to equal 2.0 ml , 
 and the sample was then vortexed until it was thoroughly mixed . samples 
 were then homogenized by bead - beating in a fastprep-24 instrument 
 ( mp biomedicals , solon , oh ) for 2 min at 6.5 m / s . homogenate was incubated 
 for 5 min at 95 c , vortexed , and centrifuged at 13  000 g for 1 min to pellet stool particles . 1.2 ml of the supernatant 
 was used to purify dna with the qiagen stool mini kit according to 
 the manufacturer s instructions . preparation of fecal dna samples for 
 pyrosequencing was carried out as previously described by the core for applied genomics and ecology 
 ( cage , university of nebraska ) . the v1v3 region of the 16s 
 rrna gene was pcr amplified using bar - coded universal primers with 
 roche-454 a or b titanium adapter sequences , shown in italics , b-8f 
 ( 5-cctatcccctgtgtgccttggcagtctcagagagtttgatcmtggctcag-3 ) and a-518r ( 5-ccatctcatccctgcgtgtctccgactcagnnnnnnnnattaccgcggctgctgg-3 ) , where 
 the n represents the 8 base barcode sequence unique to each sample . 
 to ensure the representation of bifidobacteria , bifidobacteria - specific primer b-8fbif 
 ( 5-cctatcccctgtgtgccttggcagtctcagagggttcgattctggctcag-3 ) was mixed with 
 the 8f universal primer , which has a 3 base pair mismatch , at a concentration 
 of 4:1 . the pcr mixture and sequencing was performed using 
 the 454 roche sequencing primer kit from the a end and carried out 
 using the standard protocol on a roche genome sequencer gs - flx . the v4 region of the 16s rrna gene was pcr amplified using universal 
 barcoded primers with illumina sequencing adapters , shown in italics , 
 the n represents the 8 bp barcode sequence unique to each sample , 
 and the linker is in bold , v4f ( 5-aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatctnnnnnnnngtgtgccagcmgccgcggtaa-3 ) 
 and v4rev ( 5-caagcagaagacggcatacgagatcggtctcggcattcctgctgaaccgctcttccgatctccggactachvgggtwtctaat-3 ) . pcr reactions contained 12.5 l of 2 gotaq green master 
 mix ( promega , madison , wi ) , 1.0 l of 25 mm mgcl2 , 8.5 l of water , 0.5 l of forward and reverse primers 
 ( 10 m final concentration ) , and 2.0 l of genomic dna . amplicons were 
 combined and cleaned using the qiaquick 96 pcr purification kit ( qiagen , 
 valencia , ca ) . amplicon dna concentrations were quantified using the 
 quant - it picogreen dsdna kit in 96-well microplates , and fluorescence 
 detection , composite sample mixture , and gel purification were carried 
 out as previously described . the sample 
 was sent to the university of california dna technologies core facility 
 for sequencing on an illumina genome analyzer ii sequencing platform . the qiime software package ( version 
 1.4.0 ) was used to analyze the results of both the pyrosequencing 
 and illumina sequencing runs . v1v3 
 16s rrna gene sequences from pyrosequencing reads were removed from 
 analysis if they were < 200 bp in length , contained > 3 ambiguous 
 bases , had a mean quality score < 25 , contained a homopolymer run 
 greater than 6 nt , or did not contain a primer or a barcode sequence . similar sequences were clustered into operational taxonomic units 
 ( otus ) with uclust software and minimum 
 identity of 97% . taxonomy was assigned to each otu with the ribosomal database 
 project ( rdp ) classifier , with a minimum 
 support threshold of 80% and the rdp taxonomic nomenclature . otu representatives 
 were aligned against the greengenes core set with pynast software with a minimum 
 alignment length of 150 bp and a minimum identity of 75% . illumina v4 16s rrna gene sequences were demultiplexed and 
 quality - filtered using the qiime software package as well . reads were truncated after a maximum number 
 of 3 consecutive low - quality scores . after quality trimming , reads 
 were removed from analysis if they were < 60 bp and the number of 
 ambiguous bases was > 3 . otu clustering , taxonomy assignment , phylogenetic 
 tree - building , and alignment were carried out as stated above , with 
 the exception that the minimum alignment length was 75 bp . otus with 
 sequences that numbered less than 0.01% of the total reads were filtered 
 out to reduce noise in the assignments . beta diversity 
 was estimated by computing weighted and unweighted unifrac distances 
 between samples . pearson product  moment correlation coefficients 
 were calculated in qiime between the 
 nano - hplc - chip / tof ms d / h ratios of individual hmo isomers normalized 
 to baseline intensity and the relative abundances of order - level bacterial 
 taxa . sybr green and taqman qpcr assays were performed on 
 a 7500 fast real - time 
 pcr system ( applied biosystems , carlsbad , ca ) with primers specific 
 for universal bacteria , bacteroidales , and bifidobacterium , and species - specific bifidobacterium primers for b. longum group , b. adolescentis group , b. catenulatum group , b. breve , and b. bifidum . sybr green assays 
 contained 10 l of 2 takara perfect real time master mix , 
 6 l of water , 1 l each of forward and reverse primers , 
 and 2 l of genomic dna . bifidobacterium taqman qpcr assays contained 12.5 
 l of 2 taqman universal pcr master mix ( applied biosystems ) , 
 2.5 l each of forward and reverse primers , taqman probe , 3.75 
 l of water , and 2.5 l of diluted genomic dna . b. longum group taqman assays contained 10 l 
 2 taqman universal pcr master mix ( applied biosystems ) , 1 l 
 each forward and reverse primers and taqman probe , 5 l water , 
 and 2 l genomic dna . reaction conditions and primer concentrations 
 were as described in supporting information table 
 1 . bifidobacterium genus - specific primers ( nbif389 , 5-gccttcgggttgtaaac ; 
 and nbif1018r , 5-gaccatgcacccctgtg-3 ) 
 were designed targeting a consensus alignment of 16s rrna genes from 
 all bifidobacterium sequences deposited 
 in the ribosomal database project ( rdp ) database aligned using clustalx . primer specificity 
 and taxonomic coverage were determined using primerprospector , checking against a representative subset of 
 the greengenes 16s rrna database filtered at 97% identity . samples were amplified by pcr in 50 l 
 reactions containing 5100 ng of dna template , 25 l 
 of 2 promega gotaq green master mix ( promega , madison , wi ) , 
 1 mm of mgcl2 , and 2 pmol of each primer . each pcr was 
 performed in triplicate , and the products combined prior to purification . 
 the pcr conditions consisted of an initial denaturation at 95 c 
 for 2 min , followed by 30 cycles of denaturation at 95 c for 
 1 min , annealing at 51 c for 1 min , and extension at 72 c 
 for 2 min , with a final extension at 72 c for 5 min . amplicons 
 were digested using alui and haeiii following the manufacturers 
 instructions for each enzyme . the digested dna was submitted to the 
 uc davis college of biological sciences sequencing facility for fragment 
 analysis . traces were visualized using the program peak scanner v1.0 
 ( applied biosystems ) using a baseline detection value of 10 fluorescence 
 units . peak filtration and clustering were performed with r software 
 using the ibest script suite . otu picking 
 was based on an in silico digest database generated by the virtual 
 digest tool from mica of good - quality 
 16s rrna gene sequences compiled by the ribosomal database project 
 release 10 , allowing up to 3 nucleotide mismatches 
 within 15 bp of the 5 terminus of the forward primer . fecal glycoprofiles and 
 microbial profiles were obtained for weeks 0 , 1 , 2 , and 13 . previous 
 results have shown that the glycan composition is relatively constant 
 for mature milk over a 6 month period . the amount of hmo ingested increases as milk consumption increases 
 over the first several weeks of life . figure 1 shows the fecal oligosaccharide compositions ( analyzed by maldi 
 ft - icr ms ) and bacterial populations ( analyzed by 16s rdna pyrosequencing ) 
 of infant a at the four time points . corresponding 
 monoisotopic masses and compositions of the nominal masses in figure 1 are shown in table 1 . the 
 table also shows monoisotopic masses of [ m + na ] ion , 
 the commonly observed ion in maldi ft - icr ms . exact masses and elemental 
 compositions were calculated using the nist glyco mass calculator 
 ( de leoz and stein , http://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:glycocalc ) . changes in oligosaccharide intensities and bacterial population 
 in the feces of infant a. ( a ) percent change in h / d ratios of hmos 
 in the feces of infant a at weeks 0 , 1 , 2 , and 13 . ( b ) corresponding fecal bacterial population of infant 
 a using 16s rdna pyrosequencing . the monoisotopic masses of the 
 [ m + na ] precursor ion , the commonly observed ion in maldi 
 ft - icr ms , are also shown . hex , hexose ; hexnac , n - acetylhexosamine ; fuc , fucose ; neuac , n - acetylneuraminic 
 acid . the graph in week 0 ( figure 1a ) 
 should not be interpreted as each composition 
 having the same abundances . each composition is composed 
 of several isomers , as discussed in greater detail below . infant a 
 shows a general increase of all fecal hmos from week 0 to week 1 . 
 at week 2 , the amount of fecal hmos decreases to nearly the original 
 level of week 0 . at week 13 , the fecal hmos decrease to a fraction 
 of the peak amount at week 1 . the decrease is not uniform ; there are 
 a number of compositions that increase or are unchanged at week 13 . order - level fecal populations are shown in figure 2a , and genus level , in figure 2b . multiplexed 
 pyrosequencing and illumina sequencing of infant a s samples 
 yielded 12  779 and 288  390 partial 16s rrna gene sequence 
 reads ( mean length 492 and 143 bp ) , respectively . the mean number 
 of reads per sample was 5458 for pyrosequencing and 72  098 
 for illumina . the observed early predominance of streptococcus in the gut ( figure 1b ) is consistent with previous observations in breast fed 
 infants . previously demonstrated that tested species of streptococci 
 consumed hmo poorly or not at all . during the first week the fecal 
 oligosaccharides increase ; we hypothesize that they are being provided 
 by the mother but are not being consumed by the gut microbes . the 
 amount of oligosaccharides in feces peaks during week 1 ; however , 
 by week 2 , there is a noticeable decrease in intensities of hmos corresponding 
 with a small increase in fecal bacteroides sp . population ( figure 1a , b ) . members of bacteroides and bifidobacterium sp . are known to be strong consumers of hmos . illumina 
 sequencing shows a constant presence of low levels of bifidobacterium sp . in the first 3 weeks that is 
 not detected in the pyrosequencing results . interestingly , by week 
 13 , the bacteroides are replaced by bifidobacterium sp . , which dominate the fecal bacterial 
 population , and infant a microbial population changes over the first 13 
 weeks based 
 on next - generation sequencing . ( a ) illumina and ( b ) pyrosequencing 
 profile of the v4 and the v1v3 regions of the 16s rrna gene , 
 respectively . the letters preceding the taxon are taxonomy identifiers : 
 p ( phylum ) , c ( class ) , o ( order ) , f ( family ) , and g ( genus ) . bifidobacterium - specific trflp and 
 qpcr revealed a succession of bifidobacterial species over time ( figure 3 ) . infantis ) dominated these samples from weeks 013 , 
 followed by an increase in b. adolescentis , b. bifidum , and b. 
 breve at weeks 1326 ( data not shown for weeks 
 > 13 ) . infant a microbial population changes over the first 13 weeks based 
 on qpcr and bif - trflp analyses . qpcr of ( a ) total bacteria , bifidobacteria , and bacteroidales and ( b ) species - specific bifidobacteria populations ; ( c ) bif - trflp profile of the total bifidobacteria community . the correlation of bifidobacterial 
 growth with hmo consumption 
 was confirmed by calculating pearson product  moment correlation 
 coefficients between normalized hmo d / h ratios and the relative abundances 
 of all order - level bacterial taxa detected in at least three samples . bifidobacteriales was the only clade yielding a significant , 
 negative correlation coefficient with five hmos , as shown in table 2 ( negative correlation implying that as fecal bifidobacteria 
 numbers increase , fecal hmos decrease , consistent with consumption 
 of the hmos ) . other bacteria demonstrate a significant , positive correlation , 
 indicating that as these microbes increased fecal hmos increased as 
 well ( consistent with lack of consumption of hmos by these bacteria , 
 e.g. , enterobacteriales and bacillales ) . a second infant , infant 
 b , also shows development of a saccharolytic microbiota . figure 4a , b shows the glycoprofiles ( analyzed by maldi ft - icr 
 ms ) and bacterial populations ( analyzed by 16s rdna pyrosequencing ) 
 over the infant s first 14 weeks of life , respectively . we 
 observe the fecal glycans to increase dramatically from week 0 to 
 week 1 . note that the y axis in this figure is logarithmic 
 and that the increases vary from 14-fold to > 1 million - fold . at 
 week 
 2 , there is little change in hmo abundance in the feces , whereas at 
 week 14 , all of the measured fecal hmos have decreased ( range 2-fold 
 to 7000-fold higher than baseline ) . changes in oligosaccharide intensities 
 and bacterial population 
 in the feces of infant b. ( a ) percent change in h / d ratios of hmos 
 in the feces of infant b at weeks 0 , 1 , 2 , and 14 . intensities were 
 obtained using maldi ft - icr ms in ( + ) ion mode with n ( b ) corresponding fecal 
 bacterial population of infant b using 16s rdna pyrosequencing . s 
 samples yielded 30  886 and 447  149 partial 16s rrna 
 gene sequence reads , respectively . order level fecal populations are 
 shown in figure 5a , and genus level , in figure 5b . the fecal bacterial population of this infant 
 is initially populated by nearly equal amounts of enterobacteriaceae ( primarily escherichia ) and bacteroides in week 0 . by week 1 , escherichia increases to be the dominant species 
 as the amount of bacteroides decreases . 
 at week 2 , there is little change in either the fecal microbiota or 
 the fecal hmo composition . by week 14 , bacteroidales dominate the population at 60% , and the corresponding hmo fecal 
 profile shows a decrease in intensity of nearly all hmo compositions . these results correlate with our previous in vitro observations that 
 members of bacteroidaceae , e.g. , bacteroides fragilis , are consumers of hmos . infant b microbial population changes over the first 14 
 weeks of 
 life based on next - generation sequencing . ( a ) illumina and ( b ) pyrosequencing 
 profile of the v4 region and v1v3 regions of the16s rrna gene , 
 respectively . interestingly , the drop 
 in abundances of oligosaccharide for infant 
 b is not as large as that for infant a , where the majority of bacteria 
 at week 13 was bifidobacterium sp . 
 in vitro studies using strains of both bifidobacterium 
 longum ssp . infantis ( b. infantis ) and bacteriodes thetaiotaomicron show that the former is a stronger consumer of hmo than the latter.bifidobacterium sp . populations were 
 low in infant b , detectable by illumina sequencing ( figure 5a ) but not by pyrosequencing ( figure 5b ) or qpcr ( figure 6 ) . b. longum group was the only clade detected by qpcr , 
 although higher populations of b. bifidum were detected by trflp as well as small populations of b. breve and b. adolescentis ( data not shown ) . infantb microbial population changes over the first 14 
 weeks of 
 life based on qpcr analysis . * bifidobacteria are below the limit of detection ( 1  10 16s rrna 
 genes per gram of stool ) . a more 
 extensive analysis supports the hypothesis of structure / isomer - specific 
 consumption of hmos in the infants gut . the fecal profile 
 of infant a is dominated by bifidobacterium spp . by week 13 ( figure 1 ) , and the fecal 
 profile of infant b is dominated by bacteroides spp . by week 14 ( figure 4 ) . although the 
 patterns are not equivalent , suggesting differential consumption , 
 there are some similarities in the consumption or nonconsumption of 
 certain hmos . in the fecal profile of infant a , for example , 
 a pentasaccharide with monoisotopic mass of 855.3220 having the composition 
 3 hexose ( galactose or glucose ) , 1 n - acetylglucosamine , 
 and 1 fucose was found to have four isomers corresponding to the common 
 name lacto - n - fucopentaose : lnfp i , lnfp ii , lnfp 
 iii , and lnfp v. the fecal profiles are shown in figure 7a as quantified by the h / d ratio using nano - hlpc chip / tof 
 ms data . one of these four isomers behaved unlike the other three 
 structurally very similar isomers . lnfp ii ( structure inset figure 7a , note the terminal -1,4-fucosylation ) continually 
 increased from week 0 to week 13 ( suggesting a lack of consumption 
 by intestinal bacteria ) . the other three isomers , lnfp i , iii , and 
 v , all increased in fecal intensity during week 1 but decreased dramatically 
 during week 2 and stayed very low through week 13 ( suggesting consumption ) . h / d ratios of two isomeric groups of oligosaccharides in the fecal 
 hmo profile of infant a. h / d ratios were calculated using nano - hlpc 
 chip / tof ms data . ( a ) four isomers of m / z 856 ( [ m + h ] , m = 855.3220 , second bar from the left 
 in each week in figure 1a ) . ( b ) three isomers 
 of m / z 538 ( [ m + 2h ] , z = 2 , m = 1074.3963 , fourth bar from the left 
 in figure 1a ) . figure 7b demonstrates another group of 
 isomers from infant a with an overall hexasaccharide structure with 
 a common monoisotopic mass of 1074.3963 ( m / z 538 , z = 2 ) and composed of 4 hexose 
 and 2 n - acetylglucosamine with at least three linear 
 or branched structures containing terminal (1,3)- and (1,4)-galactose 
 residues . all three isomers follow a similar profile increasing in 
 week 1 and decreasing thereafter ( consistent with consumption ) . lc  ms 
 extracted ion chromatograms of m / z 538 ( [ m + 2h ] , z = 2 , m 
 = 1074.3963 ) in the fecal hmo profile of infant a using nano - hlpc 
 chip / tof ms . chromatograms at ( a ) week 0 , ( b ) week 1 , ( c ) week 2 , 
 and ( d ) week 13 . figure 9 demonstrates marked differences 
 in individual fecal hmo structures of infant a over time , consistent 
 with the hypothesized highly selective nature of consumption of hmos 
 by the intestinal bacteria . i , mflnh 
 iii , iflnh i , and iflnh iii ( mw 1220.4542 ) ) are present in the feces 
 until week 2 but diminish rapidly by week 13 ( concurrent with the 
 bloom of bifidobacteria ) , suggesting preferential consumption ; meanwhile , 
 isomer 4120a increases at week 13 , suggesting that the fecal microbes 
 may be unable to digest this structure ( figure 9a ) . isomers dflnhb , and dflnhc ( mw 1366.512 ) both show a decrease 
 concomitant with the bloom of bifidobacteria at week 13 ; however , 
 this pattern is not seen with isomer dflnha ( figure 9b ) . isomers 5130a , 5130b , f - lno , and 5130c ( mw 1585.5864 ) 
 all diminished at week 13 , suggesting consumption ( figure 9c ) . isomers 5230a , 5230b , and dflnno ii ( mw 1731.6443 ) 
 demonstrate the pattern consistent with consumption , but dflnno i 
 is markedly increased , suggesting a lack of consumption by the fecal 
 microbes at 13 weeks ( figure 9d ) . h / d ratios 
 of four isomeric groups of oligosaccharides in the fecal 
 hmo profile of infant a. h / d ratios were calculated using nano - hlpc 
 chip / tof ms data . ( a ) five isomers of m / z 611 ( [ m + 2h ] , z = 2 , m = 1220.4542 , 
 sixth bar from the left in each week in figure 1a ) . ( b ) three isomers of m / z 684 
 ( [ m + 2h ] , z = 2 , m = 1366.5121 , seventh 
 bar from the left in figure 1a ) . ( c ) four isomers 
 of m / z 794 ( [ m + 2h ] , z = 2 , m = 1585.5864 , tenth bar from the left 
 in figure 1a ) . ( d ) five isomers of m / z 867 ( [ m + 2h ] , z = 2 , m = 1731.6443 , 11th bar from the left in figure 1a ) . isomer analyses of fecal hmos for infant b as quantified 
 by the 
 h / d ratios using nano - hplc chip / tof ms data are shown in supporting information figures 13 . fecal 
 profiles of infant b for lacto - n - fucopentaose isomers 
 ( mw 855.3220 ) are shown in supporting information 
 figure 1a . the -1,4-fucosylated lnfp ii increased slightly 
 from week 0 to week 1 and then plateaued in weeks 214 ( 5.7753 
  0.3185 ) , suggesting nonconsumption by intestinal bacteria . the other three isomers , lnfp i , iii , and v , increased dramatically 
 in fecal intensity during week 1 but decreased in intensity at week 
 14 , suggesting consumption at the fecal profile shift to bacteroides spp . at week 14 . supporting information figure 1b shows 
 fecal profiles of another group of isomers with mw 1074.3963 . all 
 three isomers , lnh , lnnh , and p - lnh , show a decrease concomitant with 
 the bloom of bacteroides at week 14 , suggesting consumption . the extracted 
 ion chromatograms of said isomers are shown in supporting information figure 2 . all isomers increased from 
 week 0 to week 2 and then decreased in week 14 , consistent with consumption . similar to infant a , the results suggest little variation between 
 -galactosidase linkage isomers in infant b. other individual 
 fecal hmo structures and isomers are shown in supporting information figure 3 . i , mflnh iii , iflnh 
 i , and iflnh iii ) decrease over time ( supporting 
 information figure 3a ) . isomers dflnh a , b , and c all show 
 a decrease at week 14 ( supporting information 
 figure 3b ) . isomers 5130a and 5130c ( mw 1585.5864 ) both show 
 a decrease at week 14 , suggesting consumption , whereas 5130b and f - lno 
 increased at week 14 ( supporting information figure 
 3c ) . isomers 5230a and dflnno i and ii ( mw 1731.6443 ) show 
 a pattern consistent with consumption , but 5230b is markedly increased , 
 suggesting a lack of consumption by the fecal microbes at 14 weeks 
 ( supporting information figure 3d ) . quantitation of milk oligosaccharides 
 is complicated 
 by the different ionization properties of the different structures . suppression effects are observed when the sample is analyzed as a 
 mixture by mass spectrometry . for example , sialylated oligosaccharides 
 are often suppressed by neutral oligosaccharides in the positive mode , 
 whereas the reverse happens in the negative mode . separation of the oligosaccharides eliminates suppression 
 effects ; however , there are still slight variations in the ionization 
 of the individual structures . in addition , the internal standards from 
 the pooled milk sample contain more structures than any individual 
 specimen , allowing the analysis of large biological diversity . the two infants studied were both healthy , breast - fed infants , 
 but the colonization of their intestinal tract differed remarkably . the fecal microbiota of infant a was initially dominated by streptococcus spp . , whereas the gut microbiota of 
 infant b was dominated by escherichia spp . these bacteria are common commensals of the birth canal and 
 are not hmo consumers , i.e. , do not have the genetic capability to 
 produce the glycosidases necessary to break down the linkages of hmo . the increase in fecal hmos in weeks 02 in both infants is 
 consistent with the hypothesized lack of hmo consumption during this 
 period . however , by week 13 , bifidobacterium spp . dominated in infant a , and levels of most fecal hmos dropped 
 dramatically . the decline in hmo intensity is significantly negatively 
 correlated to bifidobacteriales abundance 
 and positively correlated with several other bacteria , notably , enterobacteriales and bacillales . for infant b , bacteroides spp . were 
 dominant by week 14 , at which time levels of many fecal hmos were 
 decreased compared to those at weeks 1 and 2 . the enrichment 
 of bifidobacterium spp . and bacteroides spp moreover , metagenomic analysis has revealed that this enrichment 
 also corresponds with increased expression of many genes involved 
 in consumption of complex oligosaccharides . we have previously shown in vitro that bacteroides are good consumers of hmos and that b. infantis can grow with hmos as the sole carbon 
 source . in spite of the complexity of 
 this ecological niche , the in vivo data here presented match closely 
 the in vitro results for the single species analysis . these results 
 further support the concept that one function of hmos is to selectively 
 enrich a saccharolytic bacterial consortium despite the variety of 
 bacteria introduced into the infant in the early days of life . our method allows 
 simultaneous analysis of up to 75 structurally 
 elucidated oligosaccharides and the monitoring of hundreds of structures 
 simultaneously . profiles of human milk 
 across 6 months of lactation show the constant delivery of hmos with 
 minor fluctuation particularly for the smaller , more abundant oligosaccharides 
 ( degree of polymerization < 7 ) . this 
 level of detail allows the elucidation of gross glycosidic enzyme 
 actions by the microbiota , i.e. , the consumption of known structures 
 is an indicator of the presence of specific active bacterial enzymes 
 in the gut . the whole genome sequencing of b. 
 infantis revealed a number of exoglycosidases including 
 galactosidases , fucosidases , and sialidases . this high degree of enzymatic activity is not seen in several other 
 bifidobacterial species and in part explains the dominance of the b. longum group in infant a ( b. infantis is a member of the b. longum group ) . of particular interest is the catabolism of oligosaccharides containing 
 ( 1,2)-fucose . the presence of 2fl and other ( 1,2)-fucose - containing 
 hmos such as lacto - n - fucopentaose ( lnfp ) indicates 
 that the mother has at least one functional fucosyl transferase ( fut2 ) 
 allele and is therefore a secretor ( i.e. , able to express specific 
 fucosylated structures in secretions such as tears , milk , or saliva ) . in adults , secretor status is 
 associated with increased fecal bifidobacteria and with alterations in susceptibility to viral and bacterial 
 infections . the observation that these special 
 fucosylated oligosaccharides are preferentially consumed in healthy 
 infants suggests the hypothesis that certain bifidobacteria thrive 
 in a milieu that contains either milk from a secretor mom or intestinal 
 secretions from a secretor infant . this may have particular value 
 to the fragile premature infant where human milk is partially protective 
 against sepsis and necrotizing enterocolitis and nonsecretor status may predispose to these common complications . the analysis of fecal samples from two 
 infants was designed as 
 a proof - of - concept exercise to demonstrate the potential utility of 
 analyzing the fecal microbiota and fecal glycomics in tandem . the 
 small sample size was helpful in generating hypotheses , but future 
 studies with larger sample sizes would be necessary to confirm and 
 replicate the findings . analysis of milk specimens from the 
 mothers of these infants would have been helpful to confirm consistent 
 delivery of hmos over time ( demonstrated in other mothers , but not 
 in these two cases ) . inclusion of maternal dietary analysis and details 
 regarding the mothers health status ( e.g. , obesity , diabetes , 
 medications ) would have been useful . the major strength of these observations 
 is the detailed analyses performed including differentiation of isomers 
 with differing patterns of possible consumption by the intestinal 
 microbes . diagnostic testing of the stool of infants , children , and 
 adults for rapid determination of secretor status or to identify patterns 
 of hmos or bacterial enzymes suggestive of increased risk for necrotizing 
 enterocolitis , clostridium difficile colitis , or inflammatory diseases of the gut may be possible with 
 approaches similar to those demonstrated herein this 
 proof - of - concept study combines glycomics and genomics to 
 monitor the changes in hmos and gut microbiota in breastfeeding infants . the observed changes over time in two infants suggest the need for 
 more extensive studies of a larger cohort to test the hypothesis that 
 hmos enrich a cognate oligosaccharide - consuming microbial population 
 in the infant gut by orchestrating a shift in the infant fecal microbiota 
 from a nonsaccharolytic population dominated by commensals of the 
 birth canal to a population dominated by saccharolytic microbes concurrent 
 with a decrease in fecal hmo intensities . further studies in nonhuman 
 primates and other mammals would be valuable to determine whether 
 feedback evolutionary mechanisms link the infant gut and maternal 
 milk oligosaccharide production .