Strain-resolved community genomic analysis of gut
microbial colonization in a premature infant
Michael J. Morowitza,1, Vincent J. Denefb, Elizabeth K. Costelloc, Brian C. Thomasb, Valeriy Poroykoa,
David A. Relmanc,d,e, and Jillian F. Banfieldb,f,2
aDepartment of Surgery, University of Chicago Pritzker School of Medicine, Chicago, IL 60637; Departments ofbEarth and Planetary Science, and
fEnvironmental Science, Policy, and Management, University of California, Berkeley, CA 94720;cDepartment of Microbiology and Immunology,dDepartment
of Medicine, Stanford University School of Medicine, Stanford, CA 94305; andeVeteran’s Affairs Palo Alto Heath Care System, Palo Alto, CA 94304
Edited* by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved November 30, 2010 (received for review August 7, 2010)
The intestinal microbiome is a critical determinant of human
health. Alterations in its composition have been correlated with
chronic disorders, such as obesity and inflammatory bowel disease
in adults, and may be associated with neonatal necrotizing enter-
ocolitis in premature infants. Increasing evidence suggests that
strain-level genomic variation may underpin distinct ecological
trajectories within mixed populations, yet there have been few
strain-resolved analyses of genotype–phenotype connections in
the context of the human ecosystem. Here, we document strain-
level genomic divergence during the first 3 wk of life within the
fecal microbiota of an infant born at 28-wk gestation. We obser-
ved three compositional phases during colonization, and recon-
structed and intensively curated population genomic datasets
from the third phase. The relative abundance of two Citrobacter
strains sharing ~99% nucleotide identity changed significantly
over time within a community dominated by a nearly clonal Ser-
ratia population and harboring a lower abundance Enterococcus
population and multiple plasmids and bacteriophage. Modeling
of Citrobacter strain abundance suggests differences in growth
rates and host colonization patterns. We identified genotypic
variation potentially responsible for divergent strain ecologies,
including hotspots of sequence variation in regulatory genes and
intergenic regions, and in genes involved in transport, flagellar
biosynthesis, substrate metabolism, and host colonization, as well
as differences in the complements of these genes. Our results
demonstrate that a community genomic approach can elucidate
gut microbial colonization at the resolution required to discern
medically relevant strain and species population dynamics, and
hence improve our ability to diagnose and treat microbial
human microbiome|metagenomics|strain variation|succession|
of energy from dietary substrates, production of essential
nutrients, and protection against colonization by pathogens (1,
2). Although the adult gut microbiota is highly variable between
individuals, it displays limited diversity at the phylum level: only
two bacterial phyla (Bacteroidetes and Firmicutes) contribute
∼90% of all microbes (3). In infants, early assembly of the gut
microbiota has been linked to development of innate immune
responses and terminal differentiation of intestinal structures
(4). The dynamic process of colonization has been well studied at
high taxonomic levels (5) and seems predictable based on com-
petitive interactions between and within the dominant phyla (6).
Yet at lower taxonomic levels, and at early stages of develop-
ment, our knowledge of this process is incomplete.
typing (MLST) and comparative genomics have been used to dif-
ferentiate closely related organisms (7, 8). However, important
contextual information may be lost when interpreting genomic
variation between strains isolated from different communities. Mi-
ntestinal microbes influence human health through harvesting
when analyzing isolates, can rapidly give rise to the genomic varia-
tion that underpins strain differentiation (10).
Cultivation-independent genomic analyses of time-series sam-
ples provide a way to link shifts in population abundance to ge-
netic characteristics that underlie physiological traits, such as
virulence. Here, we analyzed human intestinal colonization dur-
ing the neonatal period. We conducted a 16S rRNA gene-based
survey of fecal samples collected daily during the first 3 wk of life
of a premature infant and reconstructed and manually curated
population genomic datasets for the dominant gut microor-
ganisms in the third of three colonization phases. We chose to
focus on the premature infant microbiome because, in addition to
its medical relevance, the limited number of dominant bacterial
species in the community allows for deep sequence coverage of
Results and Discussion
Study Subject. We studied fecal samples from a female infant
delivered by caesarean section at 28-wk gestation due to pre-
mature rupture of membranes. She was treated empirically with
broad-spectrum antibiotics (ampicillin/gentamicin) for the first
7 d of life but did not receive antibiotics during the remainder of
the study period. She received enteral feedings with maternal
breast milk between the fourth and ninth days of life. Feedings
were withheld between days 9 and 13 because of abdominal
distension. On day 13, feedings were slowly resumed with arti-
ficial infant formula (Similac Special Care 20 cal/fl oz; Abbott
Nutrition). She also received parenteral nutrition until caloric
intake from enteral nutrition was adequate (day 28). She had no
major illnesses during her hospitalization and was discharged to
home at 64 d of life. Fecal samples were collected daily as
available between days 5 and 21.
Day-to-Day Dynamics of Community Composition. Sequencing of
amplified bacterial 16S rRNA genes (SI Materials and Methods
and Table S1 A and B) from 15 fecal samples collected on dif-
ferent days during the first 3 wk revealed three distinct com-
munity configurations demarcated by rapid transitions. This
finding is consistent with previously reported colonization pat-
terns in term infants: relative stability over days to months
Author contributions: M.J.M., V.J.D., and J.F.B. designed research; M.J.M., V.J.D., E.K.C.,
B.C.T., V.P., and J.F.B. performed research; V.J.D., B.C.T., and J.F.B. contributed new
reagents/analytic tools; M.J.M., V.J.D., E.K.C., D.A.R., and J.F.B. analyzed data; and
M.J.M., V.J.D., E.K.C., D.A.R., and J.F.B. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
Data deposition: The sequences reported in this paper have been deposited in the Se-
quence Read Archive (accession no. SRA026959) and GenBank database.
1Present address: Department of Surgery, University of Pittsburgh Medical Center,
Pittsburgh, PA 15213.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 18, 2011
| vol. 108
| no. 3www.pnas.org/cgi/doi/10.1073/pnas.1010992108
punctuated by rapid compositional change (5, 11). Marked shifts
in abundant lineages around days 9 and 15 seemed to follow
dietary adjustments. On days 5 through 9, communities were
largely composed of Leuconostoc, Weissella, and Lactococcus
(Fig. 1A). The genera Pseudomonas and Staphylococcus, which
were relatively scarce on days 8 and 9, became abundant by day
10. On days 10 through 13, species richness and evenness were
relatively low (Table S1) and Pseudomonadaceae predominated
(Fig. 1A). After resuming feedings on day 13, taxa characteristic
of the next phase appeared (Fig. 1A). On days 16 through 21,
species richness and evenness recovered (Table S1) and the
family Enterobacteriaceae and its constituent genera Citrobacter
and Serratia came into the majority. Sample clustering based on
community-wide similarity in membership and structure (Fig. 1B
and Fig. S1 C–F) further delineated three microbiome config-
urations. Bacterial community membership and structure were
significantly more similar within, than between these colonization
phases (P < 0.001; PERMANOVA with Monte Carlo). A cross-
study comparison suggests that the infant studied here harbored
similar bacteria to those found in other premature infants sur-
veyed using equivalent methods, especially during the first and
third colonization phases (Fig. 1B) (5, 12–19).
Metagenomic Data Processing. Genome-wide sequencing of DNA
from fecal samples collected on days 10, 16, 18, and 21 yielded
245 Mbp of metagenomic sequence data. These data were
coassembled using Newbler, keeping track of each read’s sample
of origin for quantification. Quantification of community com-
position based on read abundance can be confounded by DNA
extraction and sequencing biases (20). However, we could ana-
lyze relative abundance shifts across the third colonization phase
because the same biases were expected in all samples (Fig. 2).
We identified three major sequence “bins” for Serratia, Cit-
robacter, and Enterococcus, which dominated the third phase of
colonization (Figs. 1A and 2). Projecting the smaller contig data
(500–1,500 bp) onto an emergent self-organizing map generated
based on tetranucleotide frequencies of contigs >1,500 bp and
reference genomes allowed us to assign additional fragments to
Enterococcus and provide partial coverage for one or more
Pseudomonas populations from the day 10 sample (SI Materials
and Methods and Fig. S2). Most fragments from other minor
populations were assigned to higher taxonomic levels (mostly
Enterobacteriaceae) (Table S3 in Dataset S1). We also identified
multiple plasmid and phage populations, some of which were
completely sequenced (Table S4 in Dataset S1).
Assembly of a Near-Clonal Serratia Genome and Comparative
Genomics. Manual curation resulted in a Serratia genome
(strain UC1SER) with nine gaps, seven of which involve rRNA
operons. Based on the sequence coverage of Serratia (∼17×)
compared with other bacterial contigs (Table S2), UC1SER
dominated the community genomic datasets from the formula-
fed (third) phase. We detected remarkably low levels of nucle-
otide polymorphisms in the UC1SER sequences (close to the
expected sequencing substitution error rate), and only very few
regions in which gene content varied.
Serratia, a genus comprising motile, facultative anaerobes from
the family Enterobacteriaceae, is found in many environments.
The UC1SER genome assembled de novo from metagenomic
data was compared with the publicly available genomes of Serratia
proteamaculans (21) and Serratia marcescens (Sanger Institute,
United Kingdom). S. marcescens is an important opportunistic
pathogen and a known cause of nosocomial disease in neonatal
intensive care units (22). S. proteamaculans is an endophytic bac-
genome fragments (up to 2.36 Mb in length) share a syntenous
backbone with the previously reported genomes, although nu-
merous genomic differences were noted relative to the previously
sequenced species (Table S5 in Dataset S1). For syntenous
orthologs, UC1SER predicted proteins share 97.3% average
amino acid identity (AAI) over 4,089 genes and 88.6% AAI over
across reconstructed genome fragments, we ordered the nine
UC1SER genome fragments according to the reference genomes
(Table S5 in Dataset S1).
Within syntenous regions in UC1SER, there are small clusters
of genes that occur elsewhere in S. marcescens and S. protea-
maculans. These clusters encode proteins involved in proto-
catechuate utilization, fimbrial biosynthesis and export, nitrate
and transport, tetrathionate reduction and regulation, osmopro-
tectant transport, and general metabolism, including amino acid
biosynthesis. These rearranged or “indel” regions show elevated
sequence divergence relative to syntenous orthologs (AAI of 77
and 58% relative to S. marcescens and S. proteamaculans, re-
45 samples from 9 individuals (Costello et al 2009)
3 samples from 3 individuals (Eckburg et al 2005)
2 samples from 2 individuals (Palmer et al 2007)
8 samples from 7 individuals (Palmer et al 2007)
12 samples from 12 individuals (Mshvildadze et al 2010)
11 samples from 11 individuals (Figure S8)
2 agg. samples from 20 individ. (Wang et al 2009)
15 samples from 1 individual, this study
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Day after birth
Proportion of sequences
breast milkno enteral feedingcommercial infant formulaDiet:
bacterial taxa in 15 fecal samples collected between days 5 and 21. Sequences were classified to the highest taxonomic level to which they could be con-
fidently assigned. Dots indicate metagenomic survey dates. Relevant clinical features are shown along the x axis. (B) Principal coordinates analysis of un-
weighted UniFrac distances between fecal microbiotas shown in A and those from recently published surveys of adults (3, 5, 47), term infants (5), and preterm
infants (17, 19), and from a survey of gut microbes from premature infants in a companion study (Fig. S8). Each circle corresponds to a collection of 16S rRNA
gene sequences colored according to study. Samples from this work (black circles) are labeled by day. The percentage of variation explained by the plotted
principal coordinates is indicated on the axes. Large-scale alterations in the infant’s gut microbiota composition occurred around days 9 and 15.
Multiple stable compositional states in the developing gut microbiota of the premature infant. (A) Relative abundance of the 20 most dominant
Morowitz et al. PNAS
| January 18, 2011
| vol. 108
| no. 3
spectively). Thus, these regions may contribute to metabolic var-
iation that differentiates these species.
Regions of the UC1SER genome that are absent in one or
both of the other Serratia species encode factors involved in trans-
port (most notably iron uptake) and regulation, outer membrane
and exopolysaccharide biosynthesis, adhesion, antibiotic biosyn-
thesis, virulence, quorum sensing, biosynthesis of the redox cofac-
tor pyrroloquinoline quinone, arsenate resistance, and propanoate
metabolism (Table S5 in Dataset S1). Only UC1SER contains pga
operon genes involved in polysaccharide synthesis for biofilm ad-
hesion and a regulon for allantoin utilization, which may be asso-
ciated with virulence (23). It is also the only genome with yjf-sga
operon genes (phosphotransferase system components sgaH, U,
E),whichenablesomestrainsofgutbacteriatouse vitaminC as an
biosynthesis protein not found in the other genomes. In contrast to
the other reconstructed genomes in this study, UC1SER contains
few mobile element-derived sequences.
Analyses of Two Ecologically Distinct Citrobacter Subpopulations.
Based on 16S rRNA gene sequences on assembled contigs, Cit-
robacter in the third colonization phase is closely related to Cit-
robacter freundii. Despite average coverage of ∼13× on larger
Citrobacter fragments, automated assembly resulted in a highly
fragmented genome. Citrobacter contigs displayed many diallelic
sites among their reads that were almost always linked (i.e., no
evidence for homologous recombination), indicating the pres-
ence of two coassembled strain populations. Examination of
most contig ends revealed path bifurcation (Fig. 3A) because of
local strain sequence divergence, differences in gene content,
and intergenic region length (see below).
Manual curation resolved these bifurcations and reduced the
number of Citrobacter contigs from ∼1,400 to 10 (the largest
curated contig is 2.55 Mb) (Fig. 3B). The final contigs are gen-
erally syntenous with the Citrobacter 30_2 strain draft genome
(Broad Institute, Cambridge, MA) and the complete Citrobacter
koseri ATCC BAA-895 genome (Washington University,
St. Louis, MO). Consequently, the fragments were oriented and
ordered by reference to the C. koseri genome to generate a final
genome representation for the dominant strain, UC1CIT-i
(Table S6 in Dataset S2). Of the ten genome gaps, eight are the
rRNA-encoding regions that could not be resolved, one is within
a prophage, and one is in the intergenic region between genes on
contig ends that are adjacent in both isolate genomes.
Citrobacter species are facultative anaerobes from the family
Enterobacteriaceae and are commonly found as commensals
within the mammalian intestinal tract. Like Serratia, they have
been frequently documented as pathogens in premature new-
borns (25) (e.g., in cases of neonatal meningitis). Citrobacter 30_2
was isolated from a patient with Crohn disease, whereas C. koseri
was isolated from an infant with meningitis. UC1CIT strains lack
a “supercontig” of 402 genes reported as part of Citrobacter 30_2;
based on our assembly and the functional annotation, we suspect
this supercontig derives from a megaplasmid.
As expected based upon the known physiology of human-
associated Citrobacter strains (25), the UC1CIT strains have
numerous genes for uptake and utilization of a wide variety of
substrates. Similar to C. koseri and Citrobacter 30_2, the UC1CIT
strains are predicted to express curli and fimbriae that mediate
biofilm formation and binding to host epithelial cells (26)
(Table S6 in Dataset S2). Interestingly, the UC1CIT strains and
C. koseri have dual flagellar systems but Citrobacter sp. 30_2 lacks
a lateral flagellar apparatus (Table S7 in Dataset S2). Lateral
flagella confer swarming motility in viscous fluids (e.g., mucus)
and have been associated with virulence, adhesion, and biofilm
formation (27, 28).
UC1CIT sequence variation occurs genome-wide, but one
sequence type dominates at most loci (Table S6 in Dataset S2).
Given evidence for clonal rather than recombined strains, we
UC1CIT-i strain reads
UC1CIT-ii strain reads with SNPs
12 6 3
Generation time (hr)
Colon transit time (hr)
UC1CIT-i: day 16 -> 18
UC1CIT-i: day 18 -> 21
UC1CIT-ii: day 16 -> 18
UC1CIT-ii: day 18 -> 21
*Generation time > Ctt
Citrobacter bin contig
Unknown bin contig
Enterobacteriaceae bin contig
16 18 21
Proportion of sequences
Day after birth
50100 150 200
Fraction of all Citrobacter cells
Modeled UC1CIT-i, luminal
Empirical UC1CIT-i, fecal
connecting reads (UC1CIT-i path)
connecting reads (UC1CIT-ii path)
populations. (A) Schematic representation of part of the fragmented UC1CIT
assembly. At the ends of many Citrobacter contigs (e.g., contig number
became too divergent (e.g., reads placed in contigs 697 vs. 640) or contained
completely novel sequence (reads placed in 642 vs. 696). (B) Condensing of
these paths by manual curation reduced the number of contigs, increased
average (white bar, left axis) and maximum contig length (black bars, right
(i+ii), curated UC1CIT-i and -ii strain contigs; cur (i), curated UC1CIT-i contigs).
(C) After curation, reads from the strains were grouped based on single nu-
cleotide polymorphism patterns using Strainer. (D) Shifts in proportion (of
the respective library total number of reads) of UC1CIT-i and UC1CIT-ii reads
over time. Error bar indicates SD between UC1CIT contigs. (E) A chemostat
model of the colon, only allowing for differences in growth rate, was used to
predict generationtime differences neededtoexplain theobserveddynamics
in D. (F) Simulation based on a model that incorporated intestinal wall at-
tachment fitted to the observed strain abundances when strain UC1CIT-ii had
a higher affinity α to the intestinal wall and the UC1CIT-i luminal maximum
growth rate μmax,uwas higher than the growth rate of UC1CIT-ii.
Analyses of two ecologically divergent Citrobacter UC1CIT sub-
DAY10 DAY16 DAY18 DAY21
contigs < 500 bp
Proportion of sequences
of the reads over the curated sequence bins across each library (as per-
centage of all reads in the libraries from day 10, 16, 18, and 21, respectively).
Population dynamics based on metagenomic profiling. Distribution
| www.pnas.org/cgi/doi/10.1073/pnas.1010992108Morowitz et al.
defined the minor strain type (UC1CIT-ii) by separating reads
primarily using polymorphism patterns in Strainer (29) (Fig. 3C),
which allowed for direct comparison of the two aligned strains.
UC1CIT-ii sequence blocks (up to a few kilobases in length)
share 98.5% average nucleotide identity with UC1CIT-i. In
regions of shared gene content, ∼90% of the UC1CIT-ii genome
was reconstructed. When the UC1CIT-ii strain blocks were
linked and intervening gaps filled by UC1CIT-i sequence, the
strains shared 99.1 ± 0.3% average nucleotide identity across
their genomes (Table S8 in Dataset S2). The true level of simi-
larity for orthologous sequences likely lies between these values.
Based on the relative frequency of strain-associated reads in
the combined dataset for days 10, 16, 18, and 21, UC1CIT-i
comprised 77% of the Citrobacter population (SI Materials and
Methods and Table S8 in Dataset S2). However, the relative
abundance of the strains changed dramatically during the third
colonization phase (Fig. 3D and Table S8 in Dataset S2). Pos-
sible explanations for the strain abundance shifts include: (i)
a bloom of a strain-specific phage that decimated the UC1CIT-ii
population around day 18; (ii) a reduced growth rate of
UC1CIT-ii when it was outcompeted for resources by UC1CIT-i,
Serratia or Enterococcus populations; and (iii) a higher potential
of UC1CIT-ii for intestinal wall colonization, leading to an ob-
served decrease in the luminal (fecal) population.
A phage bloom is unlikely because we did not observe an in-
crease in the abundance of Citrobacter phage sequences across
the time series. To evaluate the other hypotheses, we constructed
two models of bacterial growth in the colon (SI Materials and
Methods and Fig. S3). First, using a simplified colon chemostat
model, we calculated the differences in growth rates needed to fit
the strain population abundance shifts from days 16 to 18 and
days 18 to 21 (Fig. 3E). Assuming approximately equal numbers
of cells per milliliter luminal content, the model predicts nearly
constant generation times for UC1CIT-i. The UC1CIT-ii gen-
eration time estimates equaled those for UC1CIT-i between days
18 and 21, but increased above the colon transit time (CTT)
between days 16 and 18, resulting in washout between days 16
and 18. Based on CTT in children (12–84 h) (30) and estimates
for Escherichia coli generation times in animal models (∼2 h)
(31), results from this model guided us to select parameters for
a second model (SI Materials and Methods). The second model
incorporated intestinal wall-associated growth and enabled fit-
ting of the empirical data by assuming three orders of magnitude
higher intestinal-wall affinity for UC1CIT-ii compared with
UC1CIT-i (Fig. 3F and Fig. S3). In addition, to avoid rapid
washout of UC1CIT-i, its maximum growth rate had to be
doubled relative to UC1CIT-ii and the maximum growth rate of
wall-adherent cells had to be lowered by an order of magnitude
relative to luminal cells. Because these models were built upon
a small amount of data, they are inherently limited in their ability
to explain the Citrobacter strain behavior. However, they do
strongly suggest that the strain shifts are not the result of random
fluctuations. Regardless of whether the growth rates and in-
testinal niches differ, these Citrobacter strains are distinct in their
ability to persist in, and interact with, the human host. The
availability of genomic data for both strains provides the op-
portunity to identify possible metabolic characteristics upon
which their physiological and ecological divergence is founded.
A prominent form of variation that differentiated the two
UC1CIT strains involved insertions and deletions in intergenic
regions (Fig. 4 and Table S9 in Dataset S2). In most of the 31
observed cases, intergenic regions differed in length between the
strains by >10% and in most cases differed by ≥ 30%. Most
variable intergenic segments were flanked by gene sequences that
were nearly identical in both strains. Transcriptional regulators
[25% of cases; e.g., the LexA repressor, and the NanR regulator
of fimbrial adhesins previously shown to be affected by sequence
variation (32)] and transporters (30% of cases) were common
among the flanking genes. We identified strong predicted second-
ary structure for many divergent intergenic regions and shared
sequence similarity with known E. coli sRNAs (Fig. S4).
Hotspots of sequence variation that differentiated the UC1CIT
strains (mostly substitutions rather than sequence insertions/
deletions) also occurred within genes involved in transport, regu-
lation, motility, cell-surface composition, carbohydrate metabo-
lism, virulence, and stress response (Tables S6 and S10 in Dataset
S2). Sequence polymorphisms that could potentially affect path-
ogenicity included the misL-like gene (autotransporter), fimbrial
proteins, and a polysaccharide antigen-chain regulator. Inter-
estingly, a large gene encoding RatA, believed to promote in-
testinal colonization, was a hotspot for microdiversity and was
found to be absent in both Citrobacter sp. 30_2 and C. koseri. In
Salmonella Typhimurium, RatB (and ShdA, see below) are asso-
ciated with cecal colonization and fecal shedding, and the gene
encoding this protein exhibits strain-associated sequence varia-
tion in the form of variable-number direct repeats (33). If RatA
were associated with similar phenotypes, then sequence varia-
tion between the two strains could explain differences in niche-
partitioning and fecal abundance. We also observed unusually
high amino acid sequence divergence in lateral flagellar genes
between the UC1CIT strains, which could impact interactions with
host cell surfaces (Table S7 in Dataset S2) (34). High divergence
between the UC1CIT strains in both copies of the gene encoding
carbonic anhydrase is also notable because this gene is involved in
pH homeostasis and has been identified as a colonization factor in
some pathogens (35).
mobile elements, virulence, stress
f. UC1CIT-i vs. -ii highly diverged genes:
metabolism, e transport, LPS/OM biosynthesis
intergenic indel detailed in SOM
g. UC1CIT-i vs. -ii intergenic variation:
temporal cluster 3
temporal cluster 4
temporal cluster 5
temporal cluster 6
b,c&d. UC1CIT-ii, C. sp. 30_2 & C. koseri orthologs:
temporal cluster 2
a&e. UC1CIT-i and -ii strain paths:
temporal cluster 1 & 7
0 0.1 0.2 0.3 0.4 0.5
Fraction of all genes in Y-axis category
Other host interaction factors
figure is included as Fig. S9. (a) Outside circle represents the ten contigs of
the UC1CIT-i genome. Coloring indicates read temporal distribution clusters
of the contigs condensed during curation. Genes unique to UC1CIT-i are
generally located in areas colored in blue (Fig. S2 cluster 2, Table S11 in
Dataset S2). (b) Orthologs to UC1CIT-i in UC1CIT-ii. (c and d) Orthologs to
UC1CIT-i in Citrobacter sp. 30_2 and C. koseri. (e) UC1CIT-ii paths with gene
content not shared by UC1CIT-i, colored based on read temporal distribution
clusters (Table S12 in Dataset S2). (f) Highly divergent genes between the
UC1CIT strains, colored by functional class. (Tables S6 and S10 in Dataset S2).
(g) Intergenic regions marked by indels that differentiate the UC1CIT strains
(Table S9 in Dataset S2). (B) Summary of genomic differences between the
(A) Citrobacter UC1CIT genomic overview. A larger version of this
Morowitz et al.PNAS
| January 18, 2011
| vol. 108
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Finally, gene content differentiated the UC1CIT strains
(Tables S6, S11, and S12 in Dataset S2). Although many strain-
specific genes were clearly associated with phage, several may
confer specific metabolic traits. Potentially important genes that
were found in both UC1CIT-i and Citrobacter sp. 30_2 but not in
UC1CIT-ii encoded (i) ShdA, a large virulence protein that is
part of a pathogenicity island in Salmonella Typhimurium and
essential for successful intestinal colonization (33); (ii) the inner
membrane protein YjfL; (iii) a permease specific for transport of
products of pectinolysis (KdgT); (iv) a cluster of four proteins
involved in cyclic nucleotide metabolism; (v) fimbrial proteins;
(vi) a cluster of 13 proteins involved in phenylacetate degrada-
tion; and (vii) genes involved in lipopolysaccharide and poly-
saccharide/O antigen biosynthesis (abequose). Genes unique to
UC1CIT-ii included many fimbrial genes, and genes enabling
fructose and other sugar import, streptomycin 3 biosynthesis, and
In summary, comparative genomic analyses of the UC1CIT
strains highlight metabolic and host interaction traits with the
potential to influence strain ecology (Fig. 4B). The observation
that both regulatory genes and large intergenic regions are hot-
spots for sequence divergence indicates that one basis for phys-
iological differentiation involves gene regulation, consistent with
prior studies implicating regulation as an evolutionary mecha-
nism underlying early ecological differentiation (36, 37).
Enterococcus. The Enterococcus population increased in abun-
dance during the third phase of colonization (Figs. 1 and 2). The
16S rRNA gene sequence of strain UC1ENC (from our data) is
identical to those of several E. faecalis isolates. UC1ENC shares
98.7% AAI with E. faecalis V583 (38). We mapped the UC1ENC
contigs and reads to the V583 genome and recovered ∼81% of
the latter (Fig. S5 and Table S14 in Dataset S1). The genome size
is similar to that of E. faecalis T3 and T11 [available in high-
quality draft (8)]. Absence of multiple UC1ENC contigs covering
the same genomic region and low SNP frequency indicated that
only one strain was present (Fig. S5).
We compared the sequences of seven UC1ENC genes to
sequences of genes used in MLST analyses of clinical isolates
(http://efaecalis.mlst.net/), and found that UC1ENC was identi-
cal at all seven MLST loci to a sequence type 179, the profile of
an isolate recovered from a hospitalized patient’s blood sample
in The Netherlands. Furthermore, six out of seven loci were
identical to sequence type 16 from an isolate found in a Norwe-
gian infant’s fecal sample (39). Consistent with physiological
characterization of the latter isolate, we found genes linked to
antibiotic transport or modification and genes encoding viru-
lence factors including collagen-binding adhesin, aggregation
substance, enterococcal surface protein, gelatinase (gelE), and
cytolysin (39). Additional predicted virulence factors included an
exfoliative toxin A and a serine protease known to be transcribed
with gelE (40). Comparison with the V583 genome revealed the
absence in UC1ENC of the mobile element containing the
vancomycin resistance genes (except for vanZ), as well as small
sections of the pathogenicity island and most of the plasmid
regions and prophages (Fig. S5).
Mobile Elements and Minor Bacterial Populations. Manual curation
allowed for genomic reconstruction of a Citrobacter plasmid dis-
tinct from the above-mentioned megaplasmid of Citrobacter sp.
30_2, except for two shared regions encoding arsenate and Cu/Ag
resistance (∼85% AAI). Unlike the UC1CIT plasmid, the puta-
tive Citrobacter sp. 30_2 megaplasmid encodes tellurite resistance
genes, which have been speculated to confer protection against
mammalian host defenses (e.g., by counteracting toxic substances
produced by macrophages) (41). The UC1CIT plasmid (∼1.4
content and have read distributions across the libraries matching
the UC1CIT-i and UC1CIT-ii strains, suggesting that they are
strain-specific (Table S4 in Dataset S1). Several phage-like con-
tigs were also recovered, and some displayed boom-and-bust
dynamics, indicative of a lytic phage. We also reconstructed two
plasmids and two phage of Enterococcus with fluctuating copy
numbers (Fig. S6 and Table S4 in Dataset S1). No plasmids or
phages were linked to the Serratia population.
Low-abundance bacterial populations were genomically sam-
pled as well. As predicted by the daily 16S rRNA screening (Fig.
1), genomic sequence-abundance data suggest that Pseudomonas
peaked around day 10, whereas Enterobacter peaked on day 16,
and the Klebsiella population fluctuated over time (Fig. 2 and
Fig. S6). Several mobile elements have dynamics corresponding
to the minor Klebsiella and Enterobacter populations and may
derive from them (Fig. S6).
We performed a community-level analysis of functional po-
tential using genomic information from all populations (Fig. S7).
This analysis involved comparison of the microbiome of the
preterm infant studied here to the core human microbiome (42).
Most of the core adult orthologous groups missing from the UC1
infant communities have poorly characterized and unknown
functions. There is also a depletion of functions related to car-
bohydrate metabolism in the infant studied, perhaps because of
differences in diet and species composition, with a notable ab-
sence of lineages typical of adults from the phyla Firmicutes,
Bacteroidetes, and Actinobacteria.
Attempts to correlate gut microbial community structure with
onset of disease in premature infants have yielded conflicting
results. For example, in some studies, infants with and without
whereas in other studies samples from infants with this disease
were enriched for a particular species (e.g., Clostridium per-
fringens) or a particular phylum (e.g., Proteobacteria) (12, 19). In
a recent study, Citrobacter was detected in fecal samples from
three of four infants with NEC, but in none of the 17 control
samples (17). Although it remains possible that Citrobacter is
a causative agent for NEC, its presence in samples from the
unaffected infant in this study highlights the difficulty in con-
necting a specific bacterium to disease.
We infer from the results of this study that substantial shifts in
Citrobacter strain abundances arise as a result of strain-specific
physiology, despite a level of sequence similarity that would
typically result in classification of these species as functionally
comparable. Given the differences in genetic, especially patho-
genic, potential among the otherwise closely related Citrobacter
strains reported here, it is perhaps not surprising that medical
comparisons at the species or higher level are often inconclusive.
The intriguing differences between the UC1CIT strains in size
and sequence of a subset of intergenic regions with similarity to
small regulatory RNAs, as well as sequence divergence in regu-
latory genes, emphasize the understudied importance of the evo-
lution of gene expression in strain ecology (36).
Application of our approach to more complex microbial
communities is feasible if organisms of interest within those
communities can be deeply sampled, an objective that can be
achieved with current platforms for high throughput sequencing.
In fact, a recent study of adult gut communities that used ∼10 times
more sequencing than did our study succeeded in deeply sampling
several populations (43). Thus, ultimately, strain-resolved commu-
nity genomic approaches can provide the resolution needed for
appropriate diagnosis and treatment of a range of microbial com-
munity associated conditions.
Materials and Methods
Sample Collection. The protocol for sample collection and processing was
approved by the Institutional Review Board of The University of Chicago (IRB
#15895A). The sampling method involved manual perineal stimulation with
a lubricated cotton swab, which induced prompt defecation. Samples were
placed at −80 °C within 10 min.
Sequence Analysis of 16S rRNA Genes. Bacterial 16S rRNA genes were am-
plified using the broad-range bacterial primers 8–27F and 788–806R.
| www.pnas.org/cgi/doi/10.1073/pnas.1010992108Morowitz et al.
Sequences were processed using the QIIME software package (44) (SI Download full-text
Materials and Methods, Fig. S1, and Table S1). Fecal 16S rRNA gene
sequences from previous studies were obtained directly from GenBank or
provided by the authors. Pairwise UniFrac distances were calculated and
subjected to principal coordinates analysis (SI Materials and Methods).
Metagenomic Data Analyses. Sequencing reads from the four libraries were
coassembled using Newbler (GSassembler v. 2.0.01; Roche) after removal of
replicated reads (SI Materials and Methods). We annotated contigs larger
than 1,500 bp with an in-house annotation pipeline. Sequence bin assign-
ments were based on a combination of manual assembly curation, blastn,
blastp, GC%, sequencing depth, SNP density, and emergent self-organizing
maps (eSOM) based on tetranucleotide frequency in combination with
a K-means clustering of the temporal profiles of the reads of each contig
(SI Materials and Methods). In cases of ambiguity, contigs were assigned
to a higher phylogenetic category. Contigs of virus and plasmid origin were
primarily identified based on boom-and-bust dynamics deduced from read
temporal profiles, colocalization with plasmid/phage reference genome
fragments on the eSOM map, and functional annotation information.
Contigs between 500 and 1,500 bp were assigned to genomic bins based on
an approach similar to that used for the large contigs, except for the use of
eSOM projection. Contigs smaller than 500 nt that were not incorporated
during manual assembly curation were not further analyzed.
Assemblies for the dominant bacterial, viral, and plasmid populations
were manually curated in Consed (45). Sequences that matched the hu-
man genome (blastn e-value cutoff of 1e−35) were removed from the
dataset. For each Citrobacter contig, sequence types were identified
based on SNP patterns and separated for downstream analyses in
Strainer (29). Details on the straining process and identification of vari-
ation hotspots is described in SI Materials and Methods. Modeling of
Citrobacter strain dynamics relied on a simplified model of interstrain
competition within the colon, assuming chemostat dynamics (46) (SI
Materials and Methods and Fig. S6). The ORFs predicted on all contigs
>500 bp were contrasted to the 4,055 core adult microbiome ortholo-
gous groups by blastp analysis using the same parameters and database
used by Qin et al. (42).
ACKNOWLEDGMENTS. The authors thank the Sanger Institute for S. marces-
dataset; Dr. V. Mai for sharing 16S rRNA sequence data; Dr. C. Fischer for help
with MatLab simulations;andC.Sun, N.Justice andDr.C.Miller for comments
on the manuscript. This work was supported in part by the Surgical Infection
Society and the March of Dimes Foundation research Grant 5-FY10-103 (to
M.J.M.), Department of Energy Genomic Science program Grant DE-FG02-
05ER64134 (to J.F.B.), a Walter V. and Idun Berry Postdoctoral Fellowhip (to
and by the National Institute of Allergy and Infectious Diseases, National
Institutes of Health, and Department of Health and Human Services (Con-
tracts HHSN27220090018C and HHSN266200400001C; Broad Institute Citro-
Merigan Endowment at Stanford University.
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| January 18, 2011
| vol. 108
| no. 3