mammals are thought to develop in a bacteria - free environment within the mother 's womb .
they are born through a birth canal densely populated by lactic acid bacteria ( harrison et al . , 1953 ; dominguez - bello et al . , 2010 ; ravel et al . , 2011 ) and
, during a substantial period of their early development , feed exclusively on maternal milk .
these conserved traits may be important for the nutrition and protection of the newborn ( sela and mills , 2010 ) . in this work , we characterized the mouse intestinal microbiota from the early stages of development until weaning .
six friend leukemia virus b ( fvb ) female mice , 45 weeks old , were bred with males , and the intestinal contents of litters and mothers were sampled until weaning ( supplementary figure s1 ) .
this work was approved by the university of puerto rico iacuc ( 604 - 2008 ) .
owing to the low fecal yield in newborn mice , colon contents were collected at these stages .
we confirmed that colon contents were good proxies for feces ( supplementary figures s2s5 ) , as reported ( peterson et al .
dna was extracted from 81 samples using mobio powersoil kits ( mobio laboratories , carlsbad , ca , usa ) as recommended by the manufacturer , including bead beating .
sample dna was pcr - amplified from the variable v2 region of the 16s rrna gene , then sequenced using 454 pyrosequencing ( life sciences genome sequencer flx instrument , roche , branford , ct , usa ) as described ( andersson et al .
sequences were processed using quantitative insights into microbial ecology ( qiime ) 1.4 ( caporaso et al . , 2010 ) .
rarefaction analysis was performed based on the number of operational taxonomic units ( otus ) and the amount of phylogenetic branch length observed in each sample ( hamady et al . , 2010 ) .
beta diversity was estimated using the unifrac metric ( lozupone and knight , 2005 ) , and hierarchical clustering was performed using the unweighted pair group method with arithmetic mean ( upgma ) .
analyses of variance ( anova ) were made using the statistical program r , version 2.14.0 ( 2011 - 10 - 31 ) , using the r library .
we obtained 121  893 v2 region 16s rrna gene sequences from 81 samples , with a mean of 1505274 sequences per sample .
the global estimated coverage was 98.8% , the coverage values for the offspring at each age were as follows : day 1=96.2% day 3=98.5% day 9=99.3% day 21=97.3% adults ( older than 21 days)=96.9% .
adult mothers harbored 6121.7 fecal otus , with dominance of phyla firmicutes ( 52.3% ) , bacteriodetes ( 42.1% ) and unclassified otus ( 4.8% ) ( supplementary figure s6 ; supplementary table s1 ) .
bacterial communities of the adults did not vary significantly with age ( supplementary figure s7 ) , gender ( supplementary figure s8 ) or physiological stage associated to parturition ( anova , p=0.124 ; supplementary figure s9 ) . at day 1 after delivery
, the vaginas of the six mothers harbored 3811.5 otus , with a dominance of proteobacteria ( 80% ) and firmicutes ( 17% ) ( supplementary figure 1 ; supplementary table s2 ) .
colonic bacterial communities in newborn mice at early ages were closer to maternal communities in vaginas than to those in feces ( anova , p=0.000 ) , but by age 21 days , they clustered closer to maternal feces ( anova , p<0.001 ) , as shown by principal coordinates analysis ( supplementary figure s10 ) and upgma clustering ( supplementary figure s11 ) .
the colonic bacterial diversity decreased considerably at age 3 and 9 days ( figure 1 , supplementary figures s12s15 , supplementary table s3 ) , from nearly complete dominance of streptococcus to lactobacillus ( figure 2 , supplementary figure s16 , supplementary table s2 ) . by the time of weaning ( 21 days )
, the fecal diversity had increased to similar levels to those in the mothers ( figure 1 , supplementary figures s13s15 , supplementary table s2 ) , and with nearly complete disappearance of the streptococcus species ( figure 2 ) .
procrustes analysis shows that these results are robust to the sequence coverage obtained here ( supplementary materials ) , and the decrease in diversity during the mid - strict lactation period is supported by other diversity metrics , including phylogenetic diversity ( supplementary figure s13 ) , chao1 ( supplementary figure s14 ) and shannon entropy ( supplementary figure s15 ) .
consistent with prior studies in humans ( matsumiya et al . , 2002 ; dominguez - bello et al . , 2010 ) ,
site - specific selective factors exert pressure during development , and divergence of communities occurs in each body location .
previous work based on cultivable bacteria in mice ( schadler , 1973 ) has shown an initial colonization by lactobacilli , followed by coliforms , and finally by obligate anaerobes . in the present study , there was an initial bacterial bloom of streptococcus immediately after birth , which decreased after day 3 to be replaced by lactobacillus species that are facultative anaerobes that ferment milk lactose and casein , and produce lactic acid ( kunji et al .
, 1996 ; jiang and savaiano , 1997 ; angelakis et al . , 2012 ) .
lactate production acidifies ( ph<5.5 ) the intestinal contents and inhibits the growth of anaerobes ( soergel , 1994 ; jiang and savaiano , 1997 ) , including lachnospiracea , clostridiales and bacteroidales , whose abundance was increased by the time of weaning .
the introduction of solids to the milk diet increases the diversity of substrates for intestinal bacteria , and the strictly anaerobic colonizers become established with new pathways of fermentation , leading to the production of short - chain fatty acids , hydrogen , methane and co2 ( ruppin et al . , 1980 ) . the importance of select groups of bacteria including sfb , clostridium , bacteroides , bifidobacterium and lactobacillus , in their role on the host mucosal immune system , has been recently reviewed ( reading and kasper , 2011 ) .
contrary to the proposed developmental choreography with steady age - associated increase in microbiota alpha diversity ( koenig et al . , 2011 )
, the results presented here provide evidence of a biphasic progression toward the adult colonic microbiota , with an early reduction of diversity during suckling with dominance by lactate producers , and a second phase with increased diversity by anaerobes , coinciding with the introduction of solid food .
 the birth canal provides mammals with a primary maternal inoculum , which develops into distinctive body site - specific microbial communities post - natally . 
 we characterized the distal gut microbiota from birth to weaning in mice . 
 one - day - old mice had colonic microbiota that resembled maternal vaginal communities , but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of lactobacillus . by weaning ( 21 days ) , 
 diverse intestinal bacteria had established , including strict anaerobes . 
 our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from streptococcus to lactobacillus , followed by an increase in diversity of anaerobes , after the introduction of solid food . 
 mammalian newborns are born highly susceptible to colonization , and lactation may control microbiome assembly during early development . 
Introduction
Materials and methods
Results
Discussion
Supplementary Material