Ecological Succession of Bacterial Communities duringConventionalization of Germ-Free Mice

Merritt G. Gillilland III,a John R. Erb-Downward,a Christine M. Bassis,a Michael C. Shen,a Galen B. Toews,a Vincent B. Young,b,c

and Gary B. Huffnaglea,c

Division of Pulmonary and Critical Care Medicinea and Division of Infectious Diseases,b Department of Internal Medicine, and Department of Microbiology andImmunology,c The University of Michigan Medical School, Ann Arbor, Michigan, USA

Little is known about the dynamics of early ecological succession during experimental conventionalization of the gastrointesti-nal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jeju-num), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacte-rial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum andanalyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunumhad lower diversity and were distinct from the input community (dominated by either Escherichia or Bacteroides). However, bydays 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of thedonor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity comparedto each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is moredynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do notsimply reassemble at mucosal sites during conventionalization (they first transform into a “pioneering” community and overtime take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal envi-ronment plays a role in shaping the community.

Three processes by which the mammalian gastrointestinal (GI)tract can move from being sterile to having a mature bacterial

community are colonization following neonatal birth canal expo-sure and nursing, caesarian delivery and nursing, and experimen-tal conventionalization. Neonatal colonization occurs during de-livery when the newborn acquires a bacterial community duringtransit through the birth canal and exposure to the mother’s vag-inal microbiota (11, 18). This initial colonization is further influ-enced by interactions with the mother during suckling. Caesariandelivery results in the initial colonizing bacteria being derivedfrom the skin of the mother and other early handlers along withexposure to environmental bacteria during suckling or feeding(8). In contrast, experimental conventionalization is a processwhere an animal is born, raised, and maintained in a completelysterile environment and then given an oral in vitro-derived bacte-rial inoculum or, more commonly, a polymicrobial inoculum de-rived from another animal. For germfree mouse conventionaliza-tion, the polymicrobial inoculum often consists of cecal lumenalcontents (1, 2, 23). Conventionalization has proven to be an ex-tremely useful tool for understanding interactions between thehost and the microbiota (15, 16, 30). However, there remain gapsin our understanding of the ecological processes by which com-plex bacterial communities develop and change during transitionof the mucosa from sterility to stable colonization (20).

Developing bacterial communities in infants have been foundto be fundamentally different from the communities found in theadult GI tract (20). The structure of bacterial communities ininfants fluctuates dramatically over time, while the bacterial com-munities in adults display more stability (20). Bacterial commu-nities that form early in the neonate are frequently dominated byfacultative anaerobes such as Escherichia coli and Enterococcus spp.(4, 12, 28). These facultative anaerobes act as “pioneering species”

on the mucosa, because they consume oxygen, produce carbondioxide, alter the pH, provide additional sites for adhesion, pro-duce nutrients, and lower the redox potential, making the envi-ronment suitable for the strict anaerobes that come to dominatethe bacterial community (5, 12, 33). By 2 weeks of life, obligateanaerobes, e.g., Bacteroides spp., and Bifidobacterium spp., beginto appear (29). By 36 weeks of life, infants born vaginally begin tohave a bacterial community that resembles that of the adult gut(19); by approximately 2 to 2.5 years of age, the adult bacterialcommunity is established (7, 14).

In contrast, little is known about the dynamics of early ecolog-ical succession of bacterial communities during experimentalconventionalization of the GI tract. Ecological succession is theprocess of changes in species composition and abundance withinan ecological community across time. It has been demonstratedthat the bacterial community of the cecum of conventionalizedgermfree mice resembles the donor community (1). However,questions remain about how the structure of the bacterial com-munity is shaped during early succession, including the role ofenvironmental factors that select microbes for residency (“habitateffects”) and/or the microbial community composition in the col-onizing mixture (“legacy effects”). The goal of this study was toanalyze bacterial community dynamics (succession) and measure

Received 21 April 2011 Accepted 10 January 2012

Published ahead of print 27 January 2012

Address correspondence to Gary B. Huffnagle, ghuff@umich.edu.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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community change at two distinct sites in the intestinal tract dur-ing conventionalization of germfree mice.

MATERIALS AND METHODSMouse model and housing. The donor mouse and donor littermates wereof the conventionally raised wild-type C57BL/6 line, from the breedingcolony maintained at the University of Michigan. These mice were de-rived from mice originally purchased from Jackson Laboratories (BarHarbor, ME). All studies involving mice were approved by the UniversityCommittee on Use and Care of Animals at the University of Michigan.Germfree C57BL/6 mice were obtained from the University of MichiganGerm-Free Mouse Facility and were maintained there throughout thecourse of the experiments.

Experimental design. The cecal contents (inoculum) of a single con-ventionally raised female donor C57BL/6 mouse were processed by oralgavage into recipient female germfree mice. A total of 500 �l of cecalcontents, from the donor mouse, was diluted into 8 ml of nonpyrogenicsaline solution (0.9%) under sterile conditions, and 50 �l was processedby oral gavage into each of the recipient mice. Conventionalized micewere then harvested on days 1, 7, and 21 postinoculation. During nec-ropsy, tissue was harvested from the cecum and jejunum, rinsed in phos-phate-buffered saline to remove lumenal contents (while preserving themucosa-associated bacteria), and frozen in liquid nitrogen. Two separatedonors (donor 1 and donor 2) and their cage littermates (two for donor 1and four for donor 2) were analyzed in two independent conventionaliza-tion experiments, with a total of 5 mice/time point/donor (15 mice con-ventionalized per donor). In total, we analyzed 2 donors, 6 littermates, 10mice at day 1, 10 mice at day 7, and 10 mice at day 21 for each of two sitesin the intestine (ceum and jejunum).

DNA isolation. Genomic DNA was extracted using a Qiagen DNeasyblood & tissue kit and a modified protocol. These modifications included(i) adding a bead-beating step using UltraClean fecal DNA bead tubes(Mo Bio Laboratories, Inc.) that were shaken using a Mini-Beadbeater-8(BioSpec Products, Inc.) at the “homogenize” setting for 1 min, (ii) in-creasing the amount of buffer ATL used in the initial steps of the protocol(from 180 �l to 360 �l), (iii) increasing the volume of proteinase K used(from 20 �l to 40 �l), and (iv) decreasing the amount of buffer AE used toelute the DNA at the end of the protocol (from 200 �l to 100 �l). DNAconcentration was quantified using a NanoDrop ND-1000 Spectropho-tometer (Nanodrop Technologies).

16S Q-PCR. Quantitative PCR (Q-PCR) was used to assay thequantity of rRNA operons in the DNA samples relative to a single-copyhost gene (mouse tumor necrosis factor alpha [TNF-�]). Assays usedLightCycler 480 Probes master mix (Roche) at a 1� concentration, theappropriate primer-probe sets, and sample DNA (100 ng). For detec-tion of the bacterial signal, 2 pmol of each of the forward and reverseprimers and the fluorogenic probe was included in the reaction mix-tures. Primer and probe sequences were as follows: forward primer,5=-TCCTACGGGAGGCAGCAGT-3=; reverse primer, 5=-GGACTACCAGGGTATCTAATCTT-3=; and 16S probe, 5=-(6-carboxyfluorescein)-CGTATTACCGCGGCTGCTGGCAC-(6-carboxytetramethylrhodamine)-3=. Amplification of the bacterial signal was performed at 50°C for 2 minand 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 60 s,and a hold at 37°C. A 264-bp portion of the gene encoding TNF-� fromMus musculus was cloned and used as a positive control for the host genetarget. Detection of the host signal used 2 pmol of each of the following:forward primer (TNFa_mu_se; 5=-GGCTTTCCGAATTCAACTGGAG-3=), reverse primer (TNFa_MU_as; 5=-CCCCGGCCTTCCAAATAAA-3=), and probe (TNFa_mu_probe; 5=-Cy5-ATGTCCATTCCTGAGTTCTGCAAAGGGA-Iowa Black RQ-3=). Amplification of the host signal wasperformed at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of95°C for 20 s, 64°C for 30 s, and a hold at 37°C. Relative bacterial loadswere compared using the 2���Ct method by normalizing 16S signal to thehost TNF-� signal (27).

454 pyrosequencing and data analysis. The bacterial tag-encodedFLX-Titanium amplicon pyrosequencing (bTEFAP) method targetingthe V1-V3 hypervariable regions of the 16S rRNA gene was used to createamplicon libraries (9). Primer sets corresponded to 28F and 519R. ThebTEFAP procedures were performed at the Research and Testing Labora-tory (RTL) and followed established RTL protocols (3). Samples were alsoprocessed using the Roche 454 GS FLX Titanium platform at the Univer-sity of Michigan. For these samples, the V5-V3 hypervariable regions ofthe 16S rRNA were targeted. Primer sets corresponded to 357F and 929R,which were developed by the Broad Institute.

OTU assignment. The open-source, platform-independent, com-munity-supported software program mothur (http://www.mothur.org)(26) was used to trim, align, and cluster 16S rRNA gene sequences intooperational taxonomic units (OTUs) at a cutoff value of 0.05 and to per-form �-diversity (Shannon diversity index and Shannon evenness) and�-diversity (Morisita-Horn index) analyses. Sequence data were pro-cessed and analyzed following the Costello stool analysis example (http://www.mothur.org/wiki/Costello_stool_analysis) and the Schloss stan-dard operating procedure (SOP) (http://www.mothur.org/wiki/Schloss_SOP) referenced in mothur.

Taxonomic assignment. RDP Classifier (http://rdp.cme.msu.edu)was used for phylotyping 16S rRNA gene sequences. Sequences of lessthan 50 nucleotides and sequences without a barcode or those that had thebarcode in the wrong position were removed as low-quality reads. A con-fidence cutoff of 50% was used to produce accurate taxonomic identifi-cations (17). The number of sequences associated with each taxonomicgroup (phylum, family, or genus) in a sample was converted to an averagepercentage of community � standard error of the mean. Statistical anal-yses (analysis of variance [ANOVA] and t test) were performed usingSystat 13; a statistic was considered significant at P � 0.05.

RESULTSChanges in community diversity during conventionalization.In the first set of conventionalizations, recipient mice wereinoculated with the cecal contents of a syngeneic donor (“do-nor mouse 1”). We used 454 pyrosequencing of amplicon li-braries targeting the V1-V3 region of the 16S rRNA gene gen-erated from metagenomic cecal and jejunal mucosal samples tocharacterize the bacterial community of the cecum and jeju-num at days 1, 7, and 21 days postconventionalization. Se-quence data were binned into operational taxonomic units(OTU) and defined as sequences that were 95% similar, whichroughly approximates genus-level taxonomic classification(25). We utilized a combination of diversity metrics (Shannondiversity and Shannon evenness) to assess within-communitydiversity (�-diversity). The �-diversity of recipient cecal bac-terial communities was significantly lower on day 1 comparedto days 7 and 21 (Fig. 1) and compared to the inoculum or thelittermates of the donor. A single OTU dominated the cecalcommunity, which was identified as belonging to the genusEscherichia. Note that this OTU (Escherichia) was below detect-able levels (�0.5%) in the inoculum. The jejunum bacterialcommunity in the conventionalized mice also had significantlylower alpha diversity on day 1 compared to days 7 and 21 (Fig.1) and was dominated by the same OTU (Escherichia) that wasseen in the cecum. Comparisons of the alpha diversities of thetwo sites showed that the level was significantly higher in thececum than in the jejunum at each time point in this study.Overall, the bacterial communities in the cecum and jejunumat day 1 were less diverse and were dominated by a single OTUthat grew back from below detectable levels in the initial inoc-ulum; however, a more diverse bacterial community was estab-

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lished by day 7 and was maintained through day 21 at bothintestinal mucosal sites.

Changes in community structure and membership duringconventionalization. We next evaluated changes in bacterialcommunity structure and membership of the cecal and jejunalcommunities (�-diversity) during conventionalization. The Mori-sita-Horn similarity index is one type of metric commonly used tomeasure �-diversity, and the index values range from 0 (different)to 1 (same). The Morisita-Horn similarity indices of the cecumand inoculum were significantly lower on day 1 than on days 7 and21, and the results determined on day 7 resembled those from day21 (Fig. 2A). The same general pattern was observed for the jeju-num, although the similarity between the results determined ondays 7 and 21 decreased and none of the data from any of the timepoints were significantly different (Fig. 2A). The Morisita-Hornsimilarity indices were significantly higher in the cecum than inthe jejunum on days 7 and 21.

Additional analyses were performed to assess the similarityof the communities of the mucosal sites and also the similarityof the same sites in the littermates of the donor compared to

those of the donor. Consistent with the results observed for thecomparisons performed with the inoculum, Morisita-Hornsimilarity indices from comparisons of recipient cecum to do-nor littermate cecum versus comparisons of recipient jejunumto donor littermate jejunum were significantly lower on day 1compared to days 7 and 21 (Fig. 2B). Comparing the cecum andjejunum in the recipient mice at each time point, there was asignificant difference at day 21 but not at day 1 or 7, whichreflects changes in the jejunal but not the cecal communitybetween days 7 and 21 (Fig. 2C). This instability of the jejunalcommunities was also reflected by a significantly lower similar-ity index for comparisons of jejunal samples at all time pointscompared to comparisons of cecal samples (Fig. 2D).

Analysis of total bacterial levels by quantitative PCR of bac-terial 16S rRNA. We also measured relative bacterial colonizationlevels in the mice to determine whether the high proportion ofEscherichia at day 1 was due to outgrowth of the bacteria from theinoculum or whether it simply reflected a loss of the anaerobicpopulation. Quantitative PCR (Q-PCR) was used to quantify the16S rRNA gene levels by the 2���Ct method (27). The fold differ-ences in 16S rRNA copy numbers were determined using a single-copy host gene for normalization. As expected, there was a signif-icant difference in jejunal and cecal sample results in comparisonsof germfree mice to all other groups. In recipient mice, the bacte-rial loads in the cecum (Fig. 3A) and jejunum (Fig. 3B) were notfound to be significantly different at any of the time points or fromthose determined for the littermates of the donor. However, thelevels were different between the jejunum and cecum in both therecipient and donor littermates, with the jejunal mucosa havingfewer bacteria than the cecal mucosa. These results demonstratethat the total bacterial load in the GI tract expands rapidly uponoral inoculation into the GI tract and that the high relative pro-portion of Escherichia at day 1 reflects an initial outgrowth or“bloom” of this organism.

Taxonomic classification of the bacterial communities dur-ing conventionalization. We also analyzed the pyrosequencingdata set from the conventionalized mice by the use of RDP Clas-sifier (32) to identify changes in the bacterial community on days1, 7, and 21 and differences between intestinal sites at the sametime points. Members of three major phyla, Proteobacteria, Bacte-roidetes, and Firmicutes, were observed during conventionaliza-tion in both the cecum and the jejunum (Fig. 4A and B). Firmic-utes dominated the cecal communities of both the inoculum anddonor littermates, with the next most abundant group being theBacteroidetes. Proteobacteria were �0.5% of the community inthese donor samples. In contrast, the cecal community of recipi-ent mice on day 1 was dominated by Proteobacteria (62%). Thelevel of Proteobacteria markedly decreased between days 1 and 7and continued to decrease to nearly undetectable levels by day 21(Fig. 4A). The percentage of Firmicutes in the recipient cecal com-munity was significantly lower on day 1 than on day 7 or day 21and lower than the level seen with the littermates of the donor(Fig. 4A). While there were marked changes in the relative pro-portions of Proteobacteria and Firmicutes during the first 3 weeksof conventionalization, the levels of Bacteroidetes remained rela-tively stable throughout conventionalization, at around 15% to25% of the community.

There was a noticeable difference in the community composi-tion of the jejunum versus that of the cecum from the donorlittermates, which was partially captured during the convention-

FIG 1 Diversity (Shannon diversity index and Shannon evenness) of bacterialcommunities found in the cecum and jejunum. 454 pyrosequencing of 16SrRNA (V1-V3) was used to compare bacterial community structure and mem-bership data using operational taxonomic units (OTU). OTUs were defined at5% sequence divergence (95% similarity). Cecum and jejunum bacterial com-munities were recovered from mice inoculated with the cecal contents of do-nor mouse 1 and harvested on days 1, 7, and 21; the donor littermates (DLM)and inoculum (Inoc) are also included. n values for the groups are provided inMaterials and Methods. Bars with an asterisk represent data significantly dif-ferent from cecum day 1 data, and bars with a plus sign represent data signif-icantly different from jejunum day 1 data (Tukey’s post hoc test; P � 0.05);error bars represent standard errors of the means (SEM). (A) Mean Shannondiversity index. (B) Mean Shannon evenness.

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alization of recipients with cecal contents. The community fromthe jejunum of the donor littermates was composed of approxi-mately equal amounts of Bacteroidetes and Firmicutes, with unde-tectable levels of Proteobacteria (�0.5%; Fig. 4B). Levels of Proteo-bacteria in the recipient jejunum showed a response that wasidentical to that seen for the cecum, “blooming” at day 1 anddecreasing to background levels by day 21. However, in contrast towhat was observed in the cecal community, Proteobacteria was notthe dominant phylum in the jejunal community at day 1 (Fig. 4B).Rather, members of the Firmicutes became established by day 1and were maintained throughout the study, resulting in day 21levels that were not significantly different from those of the litter-mate jejunum. The Bacteroidetes were a minor population at day 1in the recipients but grew to a larger population by day 7 and 21and were found to be at a higher proportion in the jejunum than inthe cecum, which is similar to the results seen in the littermates ofthe donor.

Family-level classification of the bacterial communities re-vealed a high degree of similarity between those found in the re-cipient cecal mucosa (day 7 and 21), those in the donor littermatececal mucosa, and those in the inoculum. The dominant familieswere Lachnospiraceae, Porphyromonadaceae, and Ruminococ-caceae (Fig. 4C). In the cecum and jejunum of the recipient mice,the bloom of Proteobacteria at day 1 was identified as being com-

posed of Enterobacteriaceae, in particular, of the genus Escherichia,which comprised 87% of the Enterobacteriaceae identified (datanot shown); this was consistent with the conclusion from analysisof the pyrosequencing data by OTU binning prior to classification,as described above. In contrast, the jejunal mucosal communitiesof these same mice were much more heterogeneous, both betweentime points and between mice within a group, with the most con-sistently identified family being the Porphyromonadaceae (Fig.4D). Thus, at the phylum level, it would appear that convention-alization of recipients to resemble wild-type mice occurs in thejejunum; however, at a deeper taxonomic level, this does not ap-pear to be the case.

We also reanalyzed these same DNA samples, by pyrosequenc-ing, using primers targeting the V5-V3 regions of 16S rRNA andfound patterns of taxonomic diversity (see Fig. S1 in the supple-mental material) and also for Shannon’s diversity index and even-ness and Morisita-Horn index (data not shown) nearly identicalto those determined with the V1-V3 region primers. This con-firms that the results from the conventionalization were not anartifact of the 16S V region that we chose to analyze.

Visualization of the dynamics of conventionalization. Touncover underlying structural elements of the data that contrib-uted most greatly to the variance seen in the system, we analyzedthe data using principal component analysis (PCA). Samples clus-

FIG 2 �-Diversity (Morisita-Horn index) of bacterial communities found in the cecum and jejunum. 454 pyrosequencing of 16S rRNA (V1-V3) was used tocompare bacterial community structure and membership data using operational taxonomic units (OTU). OTUs were defined at 5% sequence divergence (95%similarity). Cecum and jejunum bacterial communities were recovered from mice inoculated with the cecal contents of donor mouse 1 and harvested on days 1,7, and 21; the donor littermates and inoculum are also included. The y axis represents the Morisita-Horn values, which range between 0 (not similar) and 1(same). Comparisons are statistically significant at P � 0.05; error bars represent SEM. Cec � cecum, Jej � jejunum, DLM � donor littermates, and Inoc �inoculum. (A) �-Diversity between the cecum and inoculum and the jejunum and inoculum. *, data are significantly different from cecum day 1 data; �, dataare significantly different from jejunum day 1 data. (B) �-Diversity between the cecum and DLM, jejunum and DLM, inoculum and cecum DLM, and inoculumand jejunum DLM. *, data are significantly different from cecum day 1 data; �, data are significantly different from jejunum day 1 data. (C) �-Diversity betweenthe cecum and jejunum for each time point. (D) Within-replicate �-diversity for both the cecum and jejunum. *, data are significantly different from cecum dataat same time point; �, data are significantly different from jejunum day 1 data. n values for the groups are provided in Materials and Methods.

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tered in our PCA by time and by specific anatomical site. In thececum, day 1 recipient samples clustered together, and those re-sults were separate and distinct from those determined for thececum of donor littermates. However, by day 7 and 21, the resultsfor both groups of the recipient cecum samples clustered in closeproximity to those of the donor littermates (Fig. 5A). This dem-onstrated that, over time, the cecal bacterial community of con-ventionalized mice established a community structure similar tothat in wild-type mice (donor littermates). The bacterial commu-nity of the recipient jejunum also showed considerable variation atday 1, although the amount of variation was much greater thanwas seen in cecal samples at day 1 (Fig. 5B). By day 7, the variationbetween samples had decreased and the community more closelyresembled the jejunum of the donor littermates. However, unlikethe cecum community, at day 21, the jejunal community no lon-ger clustered with the day 7 or donor littermates, suggesting thatthe bacterial community of the jejunum remained in fluxthroughout the first 21 days of conventionalization. Further anal-ysis, using PCA, revealed that it was the presence or absence oforganisms and not simply their abundance that was the predom-inant factor accounting for the differences seen in the communi-ties (see Fig. S2 in the supplemental material).

In a second set of conventionalizations, recipient mice were inoc-ulated with cecal contents from a syngeneic donor derived from aseparate litter of mice (“donor mouse 2”). A significant bloom ofProteobacteria was again evident in the cecum on day 1; however,there was also a significant bloom seen with the Bacteroidetes (Fig.4E). The particular group of Bacteroidetes to bloom was identified asbeing composed of members of the Bacteroidaceae (Bacteroides) andaccounted for nearly half of the sequences associated with this phy-lum on day 1 (Fig. 4G). By days 7 and 21, the Bacteroidetes returned tobackground levels (1 to 2% of the community), which was similar tothe level seen in the inoculum (cecal contents), donor mucosa, anddonor littermates’ mucosa. The Proteobacteria that bloomed wereEnterobacteriaceae (Escherichia) (Fig. 4G and H). The Bacteroidetesdid not bloom in the jejunum on day 1; however, there was a bloomof Proteobacteria (Fig. 4F).

PCA was used to visualize similarities in community structuresbetween groups derived from donor mouse 2. In the cecum, theday 1 communities were distinct from those of the inoculum andof the donor and donor littermates (Fig. 5C). However, by days 7and 21, the communities all clustered with the inoculum, donor,and donor littermates, demonstrating that the cecal communitieswere reconstituted after 7 to 21 days, which is similar to the resultsfrom the conventionalizations derived from donor mouse com-munity 1. The jejunal communities remained in flux throughoutthe 21 days of conventionalization and did not cluster with thececum or the jejunum of the donor or donor littermates. In thejejunums of the mice conventionalized from donor mouse 2,the pattern was again much more stochastic, and none of thegroups clustered together (Fig. 5D).

DISCUSSION

The goal of this study was to analyze the bacterial communitydynamics (succession) at two distinct sites in the intestinal tractduring conventionalization of germfree mice. We report that dur-ing the initial stages of succession, the structure and membershipof the input community was lost at both sites. However, by the endof conventionalization (day 21), the input community was suc-cessfully reconstituted in the recipient cecum, but not the jeju-num, and did not resemble that of either the cecum or the jejunumof the donor littermates. Taken together, these results reveal thatthe formation of bacterial communities during conventionaliza-tion is specific to each mucosal site, demonstrating the impor-tance of the host mucosal site in colonization.

The results of these studies demonstrate that, during conven-tionalization, “habitat effects” (environmental “filters” that selectmicrobes for residency) in a host have a greater impact on com-munity formation than do “legacy effects” (bacteria the host isexposed to). This finding is consistent with what has been previ-ously reported for transfer of the microbiota (1, 6, 24, 31), whichshowed species-level selection for a colonizing microbiome. In thestudies presented here, two distinct mucosal sites in the GI tractwithin a host exert selective pressures on the bacterial community,through yet-to-be-determined mechanisms.

At day 1 of conventionalization, the newly formed communi-ties in the cecum and jejunum were markedly different from thoseof either the inoculum or samples taken at subsequent time pointspostconventionalization. The day 1 cecal and jejunal communitiesof mice conventionalized using donor mouse 1 were dominatedby a pioneering organism (Escherichia). Escherichia is normallyfound at very low levels in the mature community in mice (be-

FIG 3 Q-PCR (2���Ct), fold copies of 16S rRNA compared to a single-copy internal control gene (TNF-�) in mice inoculated with the cecal con-tents of donor mouse 1 and harvested on days 1, 7, and 21 postinoculation;also included are day �1 (germfree controls), cecum donor littermates(DLM), and jejunum DLM. Bars with an asterisk are significantly differentfrom day �1 data (Tukey’s post hoc test; P � 0.05); error bars representSEM. (A and B) There were no significant differences in the relativeamounts of 16S rRNA (bacterial load) among inoculated groups and theDLM. Significant differences existed between the relative amount of 16SrRNA in the germfree (day �1) mice and all other groups. n values for thegroups are provided in Materials and Methods.

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FIG 4 Comparison of microbial community composition of the cecum and jejunum. 454 pyrosequencing of 16S rRNA (V1-V3 [A to D] and V5-V3 [E to H])was used to compare bacterial community structure and membership data using taxonomic-based methods (RDP Classifier). Cecum and jejunum bacterialcommunities were recovered from mice inoculated with the cecal contents of donor mouse 1 (A to D) or donor mouse 2 (E to H) and harvested on days 1, 7, and21; the inoculum, donor, and donor littermates (DLM) are also included. (A, B, E, and F) Sequences were classified at the level of phyla. The average percentageis based upon the total number of 16S rRNA sequences recovered from the community. Data from individual mice were combined for this analysis. *, **, or ***,data are significantly different from the data for the same taxonomic group on day 1 (Tukey’s post hoc test; P � 0.05); error bars represent SEM. (C, D, G, and H)Family- and genus-level diversity of bacterial communities from the cecum (C and G) and jejunum (D and H).

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tween 103 and 105 CFU/g, based on plating on violet bile agar; datanot shown). The estimated colonization levels at day 1 in the con-ventionalized recipients, compared to the inoculum, is well withinthe doubling time of 20 to 80 min that Escherichia organisms havebeen shown to achieve in vivo (10, 21–23). Early bacterial commu-nities formed during colonization in mammals are composed pri-marily of the facultative anaerobes Escherichia and Enterococcus (4,12, 13, 20, 28). After day 1, the proportion of Proteobacteria in thecommunity reassumed below-detectable levels, with the commu-nities being dominated by Firmicutes.

The community structure found in the inoculum of donor mouse2 was different from that of donor mouse 1 (Fig. 4). Even though thecommunity starting point in the inoculum was different, the out-comes of conventionalization were similar in that the final commu-nity came to closely resemble that of the donor community. Miceconventionalized via donor 2 had pioneering organisms present atday 1 (Escherichia and Bacteroides) that underwent significantblooms. Bacteroides have also been reported as early colonizers inhuman neonates (20). After day 1, the amounts of Enterobacteriaceae(Escherichia) and Bacteroidaceae (Bacteroides) returned to levels sim-ilar to those found in the inoculum, donor, and donor littermates(Fig. 4G). What both sets of these data revealed is that certain “pio-neering” organisms tended to dominate the new communities dur-ing the early stages of conventionalization.

Whether during the colonization of the neonate or the conven-tionalization of a germfree mouse, the initial environmental con-ditions, at a vacant mucosal site, are not suitable for the survival ofcertain bacterial species. The environment of the habitat initially

selects for these pioneering bacteria, and in turn these bacteriamodify the environment, making it more suitable for other bac-terial species to survive (5, 12, 33). The formation of the gut bac-terial community during conventionalization is a dynamic systemand appears facilitated by a series of changes similar to those thathappen during initial colonization.

In conclusion, conventionalization of germfree mice is highlyreproducible from mouse to mouse after mature communitieshave formed (day 7 for the cecum). Habitat effects play a crucialrole in the formation of bacterial communities during experimen-tal conventionalization of the GI tract of previously germfreemice, with less reproducibility between time points for the jeju-num when cecal contents are inoculated. However, this could be atrait of jejunal communities, in general, and not a phenomenon ofconventionalization. Most studies in mice have focused on thececum, colon, or feces; more studies on the dynamics of jejunalcommunities are warranted. More importantly, we have shownthat mature input communities do not simply reassemble at themucosal sites during conventionalization but rather first trans-form into a “pioneering” community and over time take on theappearance, in membership and structure, of the original inputcommunity. The process is analogous to that reported for neo-nates (4, 7, 14, 19, 20, 28, 29).

ACKNOWLEDGMENTS

This work was supported by NIAID grants RO1-AI064479 (G.B.H.) andR21-AI087869 (G.B.H.), NIH NIDDK grant P30DK034933 (G.B.H.),NIH grant UL1 RR024986 (the Michigan Institute for Clinical and Health

FIG 5 Operational taxonomic unit (OTU)-based ordination, using principal component analysis (PCA), of microbial communities identified in the cecum andjejunum. Cecum and jejunum bacterial communities were recovered from mice inoculated with the cecal contents of donor mouse 1 (A and B) or donor mouse2 (C and D) and harvested on days 1, 7, and 21; the inoculum, donor, and donor littermates (DLM) are also included. 454 pyrosequencing of 16S rRNA (V1-V3[A and B] and V5-V3 [C and D]) was used to compare bacterial community structure and membership data using OTUs. OTUs were defined at 5% sequencedivergence (95% similarity). Cecum (A and C) and jejunum (B and D) results are shown.

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Research), a Department of Internal Medicine pilot grant (G.B.H.), andNHLBI training grant T32HL007749 (M.G.G.).

We thank Sara Poe of the University of Michigan Germ-Free Facility;Mark Oquist for helping with the data analysis; Scot Dowd for his helpwith pyrosequencing; Patrick Schloss for his help with mothur; NicoleFalkowski and Susan Foltin, University of Michigan Microbiome Core;and Roderick McDonald, and Benjamin Murdock, Amir Sadighi Akha,and Nicole Falkowski for their review of the manuscript.

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