Ecological Succession of Bacterial Communities during ...

Ecological Succession of Bacterial Communities during Conventionalization 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 and Immunology,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 gastrointestinal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jejunum), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacterial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum and analyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunum had lower diversity and were distinct from the input community (dominated by either Escherichia or Bacteroides). However, by days 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of the donor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to 21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity compared to each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is more dynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do not simply reassemble at mucosal sites during conventionalization (they first transform into a “pioneering” community and over time take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal environment plays a role in shaping the community.

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hree 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 exposure and nursing, caesarian delivery and nursing, and experimental conventionalization. Neonatal colonization occurs during delivery when the newborn acquires a bacterial community during transit through the birth canal and exposure to the mother’s vaginal microbiota (11, 18). This initial colonization is further influenced by interactions with the mother during suckling. Caesarian delivery results in the initial colonizing bacteria being derived from the skin of the mother and other early handlers along with exposure to environmental bacteria during suckling or feeding (8). In contrast, experimental conventionalization is a process where an animal is born, raised, and maintained in a completely sterile environment and then given an oral in vitro-derived bacterial inoculum or, more commonly, a polymicrobial inoculum derived from another animal. For germfree mouse conventionalization, the polymicrobial inoculum often consists of cecal lumenal contents (1, 2, 23). Conventionalization has proven to be an extremely useful tool for understanding interactions between the host and the microbiota (15, 16, 30). However, there remain gaps in our understanding of the ecological processes by which complex bacterial communities develop and change during transition of the mucosa from sterility to stable colonization (20). Developing bacterial communities in infants have been found to be fundamentally different from the communities found in the adult GI tract (20). The structure of bacterial communities in infants fluctuates dramatically over time, while the bacterial communities in adults display more stability (20). Bacterial communities that form early in the neonate are frequently dominated by facultative anaerobes such as Escherichia coli and Enterococcus spp. (4, 12, 28). These facultative anaerobes act as “pioneering species”

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on the mucosa, because they consume oxygen, produce carbon dioxide, alter the pH, provide additional sites for adhesion, produce nutrients, and lower the redox potential, making the environment suitable for the strict anaerobes that come to dominate the bacterial community (5, 12, 33). By 2 weeks of life, obligate anaerobes, e.g., Bacteroides spp., and Bifidobacterium spp., begin to appear (29). By 36 weeks of life, infants born vaginally begin to have a bacterial community that resembles that of the adult gut (19); by approximately 2 to 2.5 years of age, the adult bacterial community is established (7, 14). In contrast, little is known about the dynamics of early ecological succession of bacterial communities during experimental conventionalization of the GI tract. Ecological succession is the process of changes in species composition and abundance within an ecological community across time. It has been demonstrated that the bacterial community of the cecum of conventionalized germfree mice resembles the donor community (1). However, questions remain about how the structure of the bacterial community is shaped during early succession, including the role of environmental factors that select microbes for residency (“habitat effects”) and/or the microbial community composition in the colonizing mixture (“legacy effects”). The goal of this study was to analyze 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, [email protected]. Supplemental material for this article may be found at http://aem.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.05239-11

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community change at two distinct sites in the intestinal tract during conventionalization of germfree mice. MATERIALS AND METHODS Mouse model and housing. The donor mouse and donor littermates were of the conventionally raised wild-type C57BL/6 line, from the breeding colony maintained at the University of Michigan. These mice were derived from mice originally purchased from Jackson Laboratories (Bar Harbor, ME). All studies involving mice were approved by the University Committee on Use and Care of Animals at the University of Michigan. Germfree C57BL/6 mice were obtained from the University of Michigan Germ-Free Mouse Facility and were maintained there throughout the course of the experiments. Experimental design. The cecal contents (inoculum) of a single conventionally raised female donor C57BL/6 mouse were processed by oral gavage into recipient female germfree mice. A total of 500 ␮l of cecal contents, from the donor mouse, was diluted into 8 ml of nonpyrogenic saline solution (0.9%) under sterile conditions, and 50 ␮l was processed by oral gavage into each of the recipient mice. Conventionalized mice were then harvested on days 1, 7, and 21 postinoculation. During necropsy, tissue was harvested from the cecum and jejunum, rinsed in phosphate-buffered saline to remove lumenal contents (while preserving the mucosa-associated bacteria), and frozen in liquid nitrogen. Two separate donors (donor 1 and donor 2) and their cage littermates (two for donor 1 and four for donor 2) were analyzed in two independent conventionalization experiments, with a total of 5 mice/time point/donor (15 mice conventionalized per donor). In total, we analyzed 2 donors, 6 littermates, 10 mice at day 1, 10 mice at day 7, and 10 mice at day 21 for each of two sites in the intestine (ceum and jejunum). DNA isolation. Genomic DNA was extracted using a Qiagen DNeasy blood & 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) increasing 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 to elute the DNA at the end of the protocol (from 200 ␮l to 100 ␮l). DNA concentration was quantified using a NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies). 16S Q-PCR. Quantitative PCR (Q-PCR) was used to assay the quantity of rRNA operons in the DNA samples relative to a single-copy host gene (mouse tumor necrosis factor alpha [TNF-␣]). Assays used LightCycler 480 Probes master mix (Roche) at a 1⫻ concentration, the appropriate primer-probe sets, and sample DNA (100 ng). For detection of the bacterial signal, 2 pmol of each of the forward and reverse primers and the fluorogenic probe was included in the reaction mixtures. Primer and probe sequences were as follows: forward primer, 5=-TCCTACGGGAGGCAGCAGT-3=; reverse primer, 5=-GGACTACCA GGGTATCTAATCTT-3=; and 16S probe, 5=-(6-carboxyfluorescein)-CG TATTACCGCGGCTGCTGGCAC-(6-carboxytetramethylrhodamine)3=. Amplification of the bacterial signal was performed at 50°C for 2 min and 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-␣ from Mus musculus was cloned and used as a positive control for the host gene target. Detection of the host signal used 2 pmol of each of the following: forward primer (TNFa_mu_se; 5=-GGCTTTCCGAATTCAACTGGAG3=), reverse primer (TNFa_MU_as; 5=-CCCCGGCCTTCCAAATAAA3=), and probe (TNFa_mu_probe; 5=-Cy5-ATGTCCATTCCTGAGTTCT GCAAAGGGA-Iowa Black RQ-3=). Amplification of the host signal was performed at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 20 s, 64°C for 30 s, and a hold at 37°C. Relative bacterial loads were compared using the 2⫺⌬⌬Ct method by normalizing 16S signal to the host TNF-␣ signal (27).

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454 pyrosequencing and data analysis. The bacterial tag-encoded FLX-Titanium amplicon pyrosequencing (bTEFAP) method targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to create amplicon libraries (9). Primer sets corresponded to 28F and 519R. The bTEFAP procedures were performed at the Research and Testing Laboratory (RTL) and followed established RTL protocols (3). Samples were also processed using the Roche 454 GS FLX Titanium platform at the University of Michigan. For these samples, the V5-V3 hypervariable regions of the 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, community-supported software program mothur (http://www.mothur.org) (26) was used to trim, align, and cluster 16S rRNA gene sequences into operational taxonomic units (OTUs) at a cutoff value of 0.05 and to perform ␣-diversity (Shannon diversity index and Shannon evenness) and ␤-diversity (Morisita-Horn index) analyses. Sequence data were processed and analyzed following the Costello stool analysis example (http: //www.mothur.org/wiki/Costello_stool_analysis) and the Schloss standard 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 less than 50 nucleotides and sequences without a barcode or those that had the barcode in the wrong position were removed as low-quality reads. A confidence cutoff of 50% was used to produce accurate taxonomic identifications (17). The number of sequences associated with each taxonomic group (phylum, family, or genus) in a sample was converted to an average percentage of community ⫾ standard error of the mean. Statistical analyses (analysis of variance [ANOVA] and t test) were performed using Systat 13; a statistic was considered significant at P ⬍ 0.05.

RESULTS

Changes in community diversity during conventionalization. In the first set of conventionalizations, recipient mice were inoculated with the cecal contents of a syngeneic donor (“donor mouse 1”). We used 454 pyrosequencing of amplicon libraries targeting the V1-V3 region of the 16S rRNA gene generated from metagenomic cecal and jejunal mucosal samples to characterize the bacterial community of the cecum and jejunum at days 1, 7, and 21 days postconventionalization. Sequence data were binned into operational taxonomic units (OTU) and defined as sequences that were 95% similar, which roughly approximates genus-level taxonomic classification (25). We utilized a combination of diversity metrics (Shannon diversity and Shannon evenness) to assess within-community diversity (␣-diversity). The ␣-diversity of recipient cecal bacterial communities was significantly lower on day 1 compared to days 7 and 21 (Fig. 1) and compared to the inoculum or the littermates of the donor. A single OTU dominated the cecal community, which was identified as belonging to the genus Escherichia. Note that this OTU (Escherichia) was below detectable levels (⬍0.5%) in the inoculum. The jejunum bacterial community in the conventionalized mice also had significantly lower alpha diversity on day 1 compared to days 7 and 21 (Fig. 1) and was dominated by the same OTU (Escherichia) that was seen in the cecum. Comparisons of the alpha diversities of the two sites showed that the level was significantly higher in the cecum than in the jejunum at each time point in this study. Overall, the bacterial communities in the cecum and jejunum at day 1 were less diverse and were dominated by a single OTU that grew back from below detectable levels in the initial inoculum; however, a more diverse bacterial community was estab-


Ecological Succession during Conventionalization

FIG 1 Diversity (Shannon diversity index and Shannon evenness) of bacterial communities found in the cecum and jejunum. 454 pyrosequencing of 16S rRNA (V1-V3) was used to compare 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 (DLM) and inoculum (Inoc) are also included. n values for the groups are provided in Materials and Methods. Bars with an asterisk represent data significantly different from cecum day 1 data, and bars with a plus sign represent data significantly 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 Shannon diversity index. (B) Mean Shannon evenness.

lished by day 7 and was maintained through day 21 at both intestinal mucosal sites. Changes in community structure and membership during conventionalization. We next evaluated changes in bacterial community structure and membership of the cecal and jejunal communities (␤-diversity) during conventionalization. The Morisita-Horn similarity index is one type of metric commonly used to measure ␤-diversity, and the index values range from 0 (different) to 1 (same). The Morisita-Horn similarity indices of the cecum and inoculum were significantly lower on day 1 than on days 7 and 21, and the results determined on day 7 resembled those from day 21 (Fig. 2A). The same general pattern was observed for the jejunum, although the similarity between the results determined on days 7 and 21 decreased and none of the data from any of the time points were significantly different (Fig. 2A). The Morisita-Horn similarity indices were significantly higher in the cecum than in the jejunum on days 7 and 21. Additional analyses were performed to assess the similarity of the communities of the mucosal sites and also the similarity of the same sites in the littermates of the donor compared to

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those of the donor. Consistent with the results observed for the comparisons performed with the inoculum, Morisita-Horn similarity indices from comparisons of recipient cecum to donor littermate cecum versus comparisons of recipient jejunum to donor littermate jejunum were significantly lower on day 1 compared to days 7 and 21 (Fig. 2B). Comparing the cecum and jejunum in the recipient mice at each time point, there was a significant difference at day 21 but not at day 1 or 7, which reflects changes in the jejunal but not the cecal community between days 7 and 21 (Fig. 2C). This instability of the jejunal communities was also reflected by a significantly lower similarity index for comparisons of jejunal samples at all time points compared to comparisons of cecal samples (Fig. 2D). Analysis of total bacterial levels by quantitative PCR of bacterial 16S rRNA. We also measured relative bacterial colonization levels in the mice to determine whether the high proportion of Escherichia at day 1 was due to outgrowth of the bacteria from the inoculum or whether it simply reflected a loss of the anaerobic population. Quantitative PCR (Q-PCR) was used to quantify the 16S rRNA gene levels by the 2⫺⌬⌬Ct method (27). The fold differences in 16S rRNA copy numbers were determined using a singlecopy host gene for normalization. As expected, there was a significant difference in jejunal and cecal sample results in comparisons of germfree mice to all other groups. In recipient mice, the bacterial loads in the cecum (Fig. 3A) and jejunum (Fig. 3B) were not found to be significantly different at any of the time points or from those determined for the littermates of the donor. However, the levels were different between the jejunum and cecum in both the recipient and donor littermates, with the jejunal mucosa having fewer bacteria than the cecal mucosa. These results demonstrate that the total bacterial load in the GI tract expands rapidly upon oral inoculation into the GI tract and that the high relative proportion of Escherichia at day 1 reflects an initial outgrowth or “bloom” of this organism. Taxonomic classification of the bacterial communities during conventionalization. We also analyzed the pyrosequencing data set from the conventionalized mice by the use of RDP Classifier (32) to identify changes in the bacterial community on days 1, 7, and 21 and differences between intestinal sites at the same time points. Members of three major phyla, Proteobacteria, Bacteroidetes, and Firmicutes, were observed during conventionalization in both the cecum and the jejunum (Fig. 4A and B). Firmicutes dominated the cecal communities of both the inoculum and donor littermates, with the next most abundant group being the Bacteroidetes. Proteobacteria were ⬍0.5% of the community in these donor samples. In contrast, the cecal community of recipient mice on day 1 was dominated by Proteobacteria (62%). The level of Proteobacteria markedly decreased between days 1 and 7 and continued to decrease to nearly undetectable levels by day 21 (Fig. 4A). The percentage of Firmicutes in the recipient cecal community was significantly lower on day 1 than on day 7 or day 21 and lower than the level seen with the littermates of the donor (Fig. 4A). While there were marked changes in the relative proportions of Proteobacteria and Firmicutes during the first 3 weeks of conventionalization, the levels of Bacteroidetes remained relatively stable throughout conventionalization, at around 15% to 25% of the community. There was a noticeable difference in the community composition of the jejunum versus that of the cecum from the donor littermates, which was partially captured during the convention-

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FIG 2 ␤-Diversity (Morisita-Horn index) of bacterial communities found in the cecum and jejunum. 454 pyrosequencing of 16S rRNA (V1-V3) was used to compare 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; ⫹, data are significantly different from jejunum day 1 data. (B) ␤-Diversity between the cecum and DLM, jejunum and DLM, inoculum and cecum DLM, and inoculum and jejunum DLM. *, data are significantly different from cecum day 1 data; ⫹, data are significantly different from jejunum day 1 data. (C) ␤-Diversity between the cecum and jejunum for each time point. (D) Within-replicate ␤-diversity for both the cecum and jejunum. *, data are significantly different from cecum data at same time point; ⫹, data are significantly different from jejunum day 1 data. n values for the groups are provided in Materials and Methods.

alization of recipients with cecal contents. The community from the jejunum of the donor littermates was composed of approximately equal amounts of Bacteroidetes and Firmicutes, with undetectable levels of Proteobacteria (⬍0.5%; Fig. 4B). Levels of Proteobacteria in the recipient jejunum showed a response that was identical to that seen for the cecum, “blooming” at day 1 and decreasing to background levels by day 21. However, in contrast to what was observed in the cecal community, Proteobacteria was not the dominant phylum in the jejunal community at day 1 (Fig. 4B). Rather, members of the Firmicutes became established by day 1 and were maintained throughout the study, resulting in day 21 levels that were not significantly different from those of the littermate jejunum. The Bacteroidetes were a minor population at day 1 in the recipients but grew to a larger population by day 7 and 21 and were found to be at a higher proportion in the jejunum than in the cecum, which is similar to the results seen in the littermates of the donor. Family-level classification of the bacterial communities revealed a high degree of similarity between those found in the recipient cecal mucosa (day 7 and 21), those in the donor littermate cecal mucosa, and those in the inoculum. The dominant families were Lachnospiraceae, Porphyromonadaceae, and Ruminococcaceae (Fig. 4C). In the cecum and jejunum of the recipient mice, the bloom of Proteobacteria at day 1 was identified as being com-

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posed of Enterobacteriaceae, in particular, of the genus Escherichia, which comprised 87% of the Enterobacteriaceae identified (data not shown); this was consistent with the conclusion from analysis of the pyrosequencing data by OTU binning prior to classification, as described above. In contrast, the jejunal mucosal communities of these same mice were much more heterogeneous, both between time points and between mice within a group, with the most consistently identified family being the Porphyromonadaceae (Fig. 4D). Thus, at the phylum level, it would appear that conventionalization of recipients to resemble wild-type mice occurs in the jejunum; however, at a deeper taxonomic level, this does not appear to be the case. We also reanalyzed these same DNA samples, by pyrosequencing, using primers targeting the V5-V3 regions of 16S rRNA and found patterns of taxonomic diversity (see Fig. S1 in the supplemental material) and also for Shannon’s diversity index and evenness and Morisita-Horn index (data not shown) nearly identical to those determined with the V1-V3 region primers. This confirms that the results from the conventionalization were not an artifact of the 16S V region that we chose to analyze. Visualization of the dynamics of conventionalization. To uncover underlying structural elements of the data that contributed most greatly to the variance seen in the system, we analyzed the data using principal component analysis (PCA). Samples clus-



FIG 3 Q-PCR (2⫺⌬⌬Ct), fold copies of 16S rRNA compared to a singlecopy internal control gene (TNF-␣) in mice inoculated with the cecal contents 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 different from day ⫺1 data (Tukey’s post hoc test; P ⬍ 0.05); error bars represent SEM. (A and B) There were no significant differences in the relative amounts of 16S rRNA (bacterial load) among inoculated groups and the DLM. Significant differences existed between the relative amount of 16S rRNA in the germfree (day ⫺1) mice and all other groups. n values for the groups are provided in Materials and Methods.

tered in our PCA by time and by specific anatomical site. In the cecum, day 1 recipient samples clustered together, and those results were separate and distinct from those determined for the cecum of donor littermates. However, by day 7 and 21, the results for both groups of the recipient cecum samples clustered in close proximity to those of the donor littermates (Fig. 5A). This demonstrated that, over time, the cecal bacterial community of conventionalized mice established a community structure similar to that in wild-type mice (donor littermates). The bacterial community of the recipient jejunum also showed considerable variation at day 1, although the amount of variation was much greater than was seen in cecal samples at day 1 (Fig. 5B). By day 7, the variation between samples had decreased and the community more closely resembled the jejunum of the donor littermates. However, unlike the cecum community, at day 21, the jejunal community no longer clustered with the day 7 or donor littermates, suggesting that the bacterial community of the jejunum remained in flux throughout the first 21 days of conventionalization. Further analysis, using PCA, revealed that it was the presence or absence of organisms and not simply their abundance that was the predominant factor accounting for the differences seen in the communities (see Fig. S2 in the supplemental material).


In a second set of conventionalizations, recipient mice were inoculated with cecal contents from a syngeneic donor derived from a separate litter of mice (“donor mouse 2”). A significant bloom of Proteobacteria 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 as being composed of members of the Bacteroidaceae (Bacteroides) and accounted for nearly half of the sequences associated with this phylum on day 1 (Fig. 4G). By days 7 and 21, the Bacteroidetes returned to background levels (1 to 2% of the community), which was similar to the level seen in the inoculum (cecal contents), donor mucosa, and donor littermates’ mucosa. The Proteobacteria that bloomed were Enterobacteriaceae (Escherichia) (Fig. 4G and H). The Bacteroidetes did not bloom in the jejunum on day 1; however, there was a bloom of Proteobacteria (Fig. 4F). PCA was used to visualize similarities in community structures between groups derived from donor mouse 2. In the cecum, the day 1 communities were distinct from those of the inoculum and of the donor and donor littermates (Fig. 5C). However, by days 7 and 21, the communities all clustered with the inoculum, donor, and donor littermates, demonstrating that the cecal communities were reconstituted after 7 to 21 days, which is similar to the results from the conventionalizations derived from donor mouse community 1. The jejunal communities remained in flux throughout the 21 days of conventionalization and did not cluster with the cecum or the jejunum of the donor or donor littermates. In the jejunums of the mice conventionalized from donor mouse 2, the pattern was again much more stochastic, and none of the groups clustered together (Fig. 5D). DISCUSSION

The goal of this study was to analyze the bacterial community dynamics (succession) at two distinct sites in the intestinal tract during conventionalization of germfree mice. We report that during the initial stages of succession, the structure and membership of the input community was lost at both sites. However, by the end of conventionalization (day 21), the input community was successfully reconstituted in the recipient cecum, but not the jejunum, and did not resemble that of either the cecum or the jejunum of the donor littermates. Taken together, these results reveal that the formation of bacterial communities during conventionalization is specific to each mucosal site, demonstrating the importance of the host mucosal site in colonization. The results of these studies demonstrate that, during conventionalization, “habitat effects” (environmental “filters” that select microbes for residency) in a host have a greater impact on community formation than do “legacy effects” (bacteria the host is exposed to). This finding is consistent with what has been previously reported for transfer of the microbiota (1, 6, 24, 31), which showed species-level selection for a colonizing microbiome. In the studies presented here, two distinct mucosal sites in the GI tract within a host exert selective pressures on the bacterial community, through yet-to-be-determined mechanisms. At day 1 of conventionalization, the newly formed communities in the cecum and jejunum were markedly different from those of either the inoculum or samples taken at subsequent time points postconventionalization. The day 1 cecal and jejunal communities of mice conventionalized using donor mouse 1 were dominated by a pioneering organism (Escherichia). Escherichia is normally found at very low levels in the mature community in mice (be-

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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 bacterial communities 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, and 21; 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 percentage is 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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FIG 5 Operational taxonomic unit (OTU)-based ordination, using principal component analysis (PCA), of microbial communities identified in the cecum and jejunum. Cecum and jejunum bacterial communities were recovered from mice inoculated with the cecal contents of donor mouse 1 (A and B) or donor mouse 2 (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% sequence divergence (95% similarity). Cecum (A and C) and jejunum (B and D) results are shown.

tween 103 and 105 CFU/g, based on plating on violet bile agar; data not shown). The estimated colonization levels at day 1 in the conventionalized recipients, compared to the inoculum, is well within the doubling time of 20 to 80 min that Escherichia organisms have been shown to achieve in vivo (10, 21–23). Early bacterial communities formed during colonization in mammals are composed primarily of the facultative anaerobes Escherichia and Enterococcus (4, 12, 13, 20, 28). After day 1, the proportion of Proteobacteria in the community reassumed below-detectable levels, with the communities being dominated by Firmicutes. The community structure found in the inoculum of donor mouse 2 was different from that of donor mouse 1 (Fig. 4). Even though the community starting point in the inoculum was different, the outcomes of conventionalization were similar in that the final community came to closely resemble that of the donor community. Mice conventionalized via donor 2 had pioneering organisms present at day 1 (Escherichia and Bacteroides) that underwent significant blooms. Bacteroides have also been reported as early colonizers in human neonates (20). After day 1, the amounts of Enterobacteriaceae (Escherichia) and Bacteroidaceae (Bacteroides) returned to levels similar to those found in the inoculum, donor, and donor littermates (Fig. 4G). What both sets of these data revealed is that certain “pioneering” organisms tended to dominate the new communities during the early stages of conventionalization. Whether during the colonization of the neonate or the conventionalization of a germfree mouse, the initial environmental conditions, at a vacant mucosal site, are not suitable for the survival of certain bacterial species. The environment of the habitat initially


selects for these pioneering bacteria, and in turn these bacteria modify the environment, making it more suitable for other bacterial species to survive (5, 12, 33). The formation of the gut bacterial community during conventionalization is a dynamic system and appears facilitated by a series of changes similar to those that happen during initial colonization. In conclusion, conventionalization of germfree mice is highly reproducible from mouse to mouse after mature communities have formed (day 7 for the cecum). Habitat effects play a crucial role in the formation of bacterial communities during experimental conventionalization of the GI tract of previously germfree mice, with less reproducibility between time points for the jejunum when cecal contents are inoculated. However, this could be a trait of jejunal communities, in general, and not a phenomenon of conventionalization. Most studies in mice have focused on the cecum, colon, or feces; more studies on the dynamics of jejunal communities are warranted. More importantly, we have shown that mature input communities do not simply reassemble at the mucosal sites during conventionalization but rather first transform into a “pioneering” community and over time take on the appearance, in membership and structure, of the original input community. The process is analogous to that reported for neonates (4, 7, 14, 19, 20, 28, 29). ACKNOWLEDGMENTS This work was supported by NIAID grants RO1-AI064479 (G.B.H.) and R21-AI087869 (G.B.H.), NIH NIDDK grant P30DK034933 (G.B.H.), NIH grant UL1 RR024986 (the Michigan Institute for Clinical and Health

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Research), a Department of Internal Medicine pilot grant (G.B.H.), and NHLBI 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 help with pyrosequencing; Patrick Schloss for his help with mothur; Nicole Falkowski 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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