JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2009, p. 1181–1189
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 4
Heterogeneity of Vaginal Microbial Communities within Individuals?#
Tae Kyung Kim,1¶† Susan M. Thomas,1¶ Mengfei Ho,2¶ Shobha Sharma,1‡ Claudia I. Reich,2
Jeremy A. Frank,2Kathleen M. Yeater,1§ Diana R. Biggs,7Noriko Nakamura,3
Rebecca Stumpf,1,5Steven R. Leigh,1,5Richard I. Tapping,1,2,6Steven R. Blanke,1,2
James M. Slauch,1,2,6H. Rex Gaskins,1,3,4Jon S. Weisbaum,6,7,8
Gary J. Olsen,1,2Lois L. Hoyer,1,4and Brenda A. Wilson1,2*
Host-Microbe Systems Theme, Institute for Genomic Biology,1Department of Microbiology,2Department of Animal Sciences,3
Department of Pathobiology,4Department of Anthropology,5and College of Medicine,6University of Illinois at
Urbana-Champaign, Carle Clinic Association,7and Carle Foundation Hospital,8Urbana, Illinois 61801
Received 4 May 2008/Returned for modification 29 July 2008/Accepted 13 January 2009
Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly
complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal micro-
bial populations have not been investigated using molecular methods. Here, we evaluated site-specific micro-
bial composition within the vaginal ecosystem and examined the influence of sampling technique in detection
of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different
locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the
vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is
not homogenous throughout the vaginal tract but differs significantly within an individual with regard to
anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal
ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical
predictor of vaginal health.
The vaginal microbiota is important for maintaining vaginal
health and preventing infections of the reproductive tract (10,
25, 34). However, studies using 16S rRNA gene clone libraries
for identifying vaginal microbes have revealed considerably
more diversity in the vaginal microbial communities of healthy
premenopausal women than previously realized (14, 17, 31, 33,
35, 36), thereby calling into question currently existing models
for a healthy vaginal ecosystem and how it might be assessed.
Although the vaginal microbiota in healthy individuals was
traditionally thought to be dominated by Lactobacillus species,
more recent studies have demonstrated that Lactobacillus is
not the predominant bacterial genus within the vaginal tracts
of a significant number of healthy women (1, 17, 31, 35, 36).
Although 60 to 70% of the women had Lactobacillus-domi-
nated vaginal microbiota, there were also individuals who
lacked Lactobacillus altogether and instead had Gardnerella,
Atopobium, Prevotella, Pseudomonas, or Streptococcus as the
predominant bacteria in the vagina (1, 17, 31, 35, 36). The
significant bacterial diversity observed among these individuals
suggests that defining a healthy vaginal environment is more
complex than originally thought.
Although the most common method of collecting vaginal sam-
ples for clinical analysis is to swab the middle or deep vaginal
canal (6, 14, 31, 35), the extent to which this approach yields
samples representative of the entire vaginal microbiota is unclear.
Indeed, the size and anatomical complexity of the vaginal tract
suggest the possibility that distinct microbial populations may
reside at discrete sites (e.g., cervix, fornix, outer vaginal canal).
However, within individuals, the presence or absence of vaginal
microniches capable of supporting discrete microbial populations
has not been evaluated. Differences in microbial population dis-
tributions within individuals may impede identification of the
specific bacterium or group of bacteria responsible for vaginal
health, which ultimately could impact clinical diagnostic evalua-
tion and treatment of vaginal disease.
To evaluate the importance of considering both sampling site
and sampling method for capturing representative vaginal micro-
bial communities within individuals, we analyzed samples ob-
tained from three different sites (cervix, fornix, outer vaginal ca-
nal) and by three different collection methods (swabbing,
scraping, lavaging) from the vaginal tracts of eight clinically
healthy (asymptomatic) women of reproductive age. We report
here that the distribution of microbiota within the human vagina
is not uniform but, instead, is distributed in a heterogeneous
MATERIALS AND METHODS
Study participants and sample collection. The Institutional Review Boards of
the University of Illinois at Urbana-Champaign and the Carle Foundation Hos-
* Corresponding author. Mailing address: Host-Microbe Systems
Theme, C3408/MC-195, Institute for Genomic Biology, University of
Illinois at Urbana-Champaign, 1206 West Gregory Drive, Urbana, IL
61801. Phone: (217) 244-9631. Fax: (217) 244-6697. E-mail: bawilson
¶ These authors contributed equally to this work.
† Present address: Department of Pharmacology, University of Penn-
sylvania, 36th and Hamilton Walk, Philadelphia, PA 19104.
‡ Present address: National Center for Biotechnology Information,
National Institutes of Health, 45 Center Drive, MSC 6510, Building 45,
Bethesda, MD 20892-6510.
§ Present address: USDA-ARS-SPA, 3810 4th Street, Lubbock, TX
# Supplemental material for this article may be found at http://jcm
?Published ahead of print on 21 January 2009.
at UNIVERSITY OF ILLINOIS on June 8, 2009
pital approved this study. Eight healthy (asymptomatic), premenopausal women
between the ages of 20 and 45 years were recruited for this study. Informed
consent was obtained from all study participants. Individuals who were asymp-
tomatic and showed no clinical signs of vaginal disease upon examination by a
physician, including evidence of vaginal discharge, amine or fishy odor, and a
vaginal pH of ?4.5, were included in the study. Individuals were not eligible for
the study if they were pregnant, lactating, menstruating, or had used antibiotics
or spermicides within 2 months prior to specimen collection. Six vaginal samples
were collected from each subject (Fig. 1). Swab samples were obtained from the
cervix, fornix, and lower one-third outer region of the vaginal canal by using
Copan sterile plastic Dacron transport swabs (HealthLink, Jacksonville, FL).
This was followed by collection of an ectocervical lavage sample using a 15-ml
sterile saline solution (Health Care Logistics, Circleville, OH). Two vaginal
scrapings were obtained from the upper (proximal) one-third and lower (distal)
one-third regions of the vaginal canal by using sterile Pap-Perfect plastic spatulas
(Medscand, Trumbull, CT), which allowed for collection of both loose and
more-adherent bacteria. All vaginal swab and scrape samples were placed in 1 ml
sterile saline (Health Care Logistics, Circleville, OH) and frozen immediately
upon collection and stored at ?80°C for up to 3 years until used. To ensure that
the microbial community profiles would be generated only from sites that had
not been previously perturbed by swab or scrape procedures, a single sample was
collected from each site, using the swab or scrape method. The one exception was
the lower one-third region of the vaginal canal, which was swabbed first and then
scraped. Vaginal smears were also obtained for each subject at the time of
clinical examination by rolling a swab across and along the length of the vaginal
wall and then onto a glass slide, which was stored at ?80°C until Gram stained
for evaluation according to the Nugent criteria (22), where a score of 0 to 3
corresponded to normal vaginal microbiota, a score of 4 to 6 was interpreted as
disturbed vaginal microbiota corresponding to an intermediate state, and a score
of 7 to 10 was classified as being consistent with bacterial vaginosis (BV). In
addition, clinical diagnostic testing for sexually transmitted diseases was per-
formed using the in-house laboratory services provided by the Carle Foundation
Hospital, including evaluation for hepatitis B virus surface antigen, human im-
munodeficiency virus type 1 (HIV-1)/HIV-2 antibody, syphilis (rapid plasma
reagin, qualitative test), Chlamydia, and Neisseria gonorrhoeae.
Genomic DNA extraction and PCR amplification. Genomic DNA was isolated
from 0.5-ml aliquots of each of the vaginal samples. To each sample, 125 ?l of 0.5
M Na-EDTA (pH 8.0) containing 75 mg/ml lysozyme (Sigma-Aldrich, St. Louis,
MO) was added, and the samples were incubated at 37°C for 30 min. To each
sample, 70 ?l of 10% sodium dodecyl sulfate and 5 ?l of 10 mg/ml proteinase K
(Fisher Scientific, Pittsburgh, PA) were added, and the resulting mixture was
subjected to three successive freeze-thaw cycles, each consisting of incubation
within a dry ice/ethanol bath for 5 min, followed by incubation at 37°C for 5 min.
The samples were then incubated at 55°C for an additional 30 min to complete
the proteinase K digestion of proteins in the disrupted cell suspension. Samples
were centrifuged at 16,000 ? g for 20 min following the addition of 70 ?l of 5 M
NaCl to the mixture and incubation on ice for 30 min. The samples were
extracted sequentially with equal volumes of phenol and phenol-chloroform-
isoamyl alcohol (Sigma-Aldrich, St. Louis, MO), followed by ethanol precipita-
tion of the genomic DNA. Dried DNA pellets were resuspended in 100 ?l of
Tris-EDTA (10 mM Tris-Cl, 1 mM EDTA [pH 8.0]) and stored at ?80°C until
used. The quality and quantity of the extracted DNA were estimated by agarose
gel analysis, using 5 ?l of the extracted DNA.
PCR amplification of the nearly full-length 16S rRNA gene was performed
using our optimized primer set, which we have previously shown to be superior
for assessing vaginal samples, particularly for bacteria such as Gardnerella and
Chlamydiales (13). This primer set was a combination of degenerate and species-
specific 27f (numbering based on Escherichia coli 16S rRNA gene positions)
forward primers (27f-YM, 27f-Bif, 27f-Bor, and 27f-Chl primers in a 4:1:1:1 ratio,
respectively) (27f-YM, 5?-AGAGTTTGATYMTGGCTCAG-3? [fourfold de-
generate universal bacterial]; 27f-Chl, 5?-AGAATTTGATCTTGGTTCAG-3?
[Chlamydiales-specific]; 27f-Bor, 5?-AGAGTTTGATCCTGGCTTAG-3? [Borre-
lia specific]; 27f-Bif, 5?-AGGGTTCGATTCTGGCTCAG-3? [Bifidobacterium
specific]) in conjunction with the 1492r bacterial reverse primer (1492r, 5?-TAC
CTTGTTACGACTT-3?). Primers were purchased from Integrated DNA Tech-
nologies (Coralville, IA). Briefly, 1 to 5 ng of total DNA was added to a PCR
mixture containing 1? platinum Taq PCR buffer (Invitrogen, Carlsbad, CA), 2
mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.2 ?M of the 27f-
YM?3 primer mixture, 0.2 ?M of the 1492r primer, and 0.25 U of platinum Taq
in a total volume of 25 ?l. PCR amplification was performed in the thermal
cycler (DNA engine PTC-200; Bio-Rad, Hercules, CA) as follows: initial dena-
turation at 94°C for 4 min, followed by 24 cycles of 94°C for 1 min, 48°C for 30 s,
and 72°C for 2 min. Upon completion of cycling, an additional 25 ?l of a PCR
mixture containing 1? platinum Taq buffer, 2 mM MgCl2, 0.2 mM deoxynucleo-
side triphosphates, 0.9 ?M of the 27f-YM?3 primer mixture, 0.9 ?M of the
1492r primer, and 0.25 U of platinum Taq was added to each PCR mixture. One
additional cycle of amplification was performed as follows: 94°C for 1 min, 48°C
for 30 s, and 72°C for 12 min. Amplification products were first purified using a
PCR purification kit (Qiagen, Valencia, CA) and then further purified using 1%
agarose gel for separation, followed by extraction using a QIAquick gel extrac-
tion kit (Qiagen, Valencia, CA).
16S rRNA gene clone library construction and sequencing. Purified PCR
products were cloned into the pCR2.1-TOPO vector and electroporated into
electrocompetent Escherichia coli TOP10 cells by using the TOPO-TA cloning
kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommenda-
tions. For each sample, at least one 96-well plate containing a library with ?90%
of the colonies carrying cloned inserts, as determined by PCR analysis, was
sequenced using the T7 promoter and M13 reverse primer sites at the high-
throughput sequencing facility of the W. M. Keck Center for Comparative and
Functional Genomics at University of Illinois at Urbana-Champaign (UIUC).
For two subjects (no. 403 and 409), an additional, independent library for each
of the six samples per subject was constructed and sequenced. The same ex-
tracted genomic DNA from each of the six samples from the two subjects was
used as the starting material for the construction of the repeat libraries. All
procedures were identical to those used for the original library construction.
Sequence processing and analysis. Sequence processing was carried out using
a series of Unix shell scripts. Forward and reverse reads from each clone were
aligned using the BL2SEQ program in the stand-alone BLAST package from
NCBI and then joined if the overlapping sequence was larger than 30 bp. For
those pairs of reads that had less than 30 bp or no overlap and thus could not be
aligned, each pair of reads was joined with five n’s between the forward and
reverse reads from each clone. These pseudojoined sequences were then sub-
jected to a BLASTN search against all joined sequences obtained from the same
clone library. If by aligning with a sequence from another clone it was revealed
that the sequences still had overlap, then the two segments were joined appro-
priately. If the two matched segments had no overlap but each read could be
matched to another clone sequence, additional n’s were added to fill the gap
between the segments based on the alignment with the matched sequence. In all
cases, vector and primer sequences were removed from the joined clone se-
quences by using the BLASTN program with parameters similar to those of the
VecScreen program from NCBI. Using this process, 4,555 sequences without
gaps and 836 sequences with gaps were obtained.
A total of 5,391 sequences were submitted to the Seqmatch program in the
Ribosomal Database Project II (RDP II) to identify related bacterial 16S rRNA
gene sequences (7). A total of 5,340 clone sequences, which had related bacterial
16S rRNA gene sequences in the RDP II database with high Seqmatch scores
(?0.8), were selected and used for further analysis. The mean Seqmatch score
was 0.97. Analysis of the 51 clone sequences, which did not match and were thus
not used for further analysis, revealed that nearly all of them were mixed clones
and probably not unique or unknown phylotypes. These 5,340 clone sequences
were again submitted to the Seqmatch program in RDP II to identify type strain
sequences most closely related to each clone. Based on the Seqmatch results, the
clone sequences could be assigned to 67 type strains (i.e., 67 phylotypes) in the
RDP II. The 16S rRNA gene sequences of the 67 type strains from RDP II and
the 5,340 clone sequences from our libraries were aligned using ClustalX (ver-
sion 1.83) or Fast Aligner in the ARB package (20, 30). The PHYLIP package
FIG. 1. Schematic representation of vaginal sampling locations.
Three swab samples were collected, from the fornix, cervix, and lower
one-third (outer) region of the vaginal canal. Two vaginal scrapings
were collected, from the upper one-third and the lower one-third of
the vagina. An ectocervical lavage sample was obtained by washing the
vaginal canal with sterile saline solution.
1182KIM ET AL. J. CLIN. MICROBIOL.
at UNIVERSITY OF ILLINOIS on June 8, 2009
was used to construct a distance matrix from the aligned 67 type strains using
DNADIST (Kimura 2 correction), and a phylogenetic tree was generated using
FITCH (the Fitch-Margoliash method) (11; http://evolution.genetics.washington
.edu/phylip.html). For bootstrap values, three methods (Fitch, parsimony, and
maximum likelihood) with 100 resamplings were performed using FITCH,
DNAPARS, DNAMLK, SEQBOOT, and CONSENSE in PHYLIP.
Statistical analysis. We used Bray-Curtis distance analysis to determine the
differences in microbial population profiles. The Bray-Curtis dissimilarity index
(5, 12), which is equivalent to a doubly weighted form of the Sørensen-Dice
dissimilarity index, was calculated according to the equation
djk? 100 ??
djk? 100 ??
when?ixij ? 1 and?ixik ? 1, where djkis the dissimilarity or distance
between communities j and k, and xijand xikare the fractional or percentage
populations of component i found in communities j and k. This distance was used
to calculate the distance matrix for cluster analysis using the unweighted-pair
group method using average linkages in the PHYLIP package.
Phylotype diversity of the combined vaginal libraries within each subject was
estimated by Simpson’s reciprocal and Shannon-Wiener’s diversity indices, using
the EstimateS software (http://purl.oclc.org/estimates). The reciprocal of Simp-
son’s diversity index (D), which provides information about the number of dis-
tinct phylotypes present in a sample, was calculated using the formula D?1?
(?pi2)?1, where piis the proportion of the ith species. The Shannon-Wiener
diversity index (H), which provides information about the community composi-
tion both in terms of number and relative abundance of distinct phylotypes, was
calculated using the formula H ? ??piln (pi), where piis the proportion of the
To determine whether the observed differences in the microbiotas between
libraries were statistically significant, the clone libraries of the six samples from
each of the eight subjects were compared using the ?-LIBSHUFF program (24).
DNA distance matrices of the 5,340 clone sequences were calculated in ARB
with the Jukes-Cantor correction (20) and subjected to ?-LIBSHUFF analysis to
compute the significance of the differences between libraries.
Bray-Curtis distance ratios. To evaluate the significance of the differences
found among the libraries from different sample types, we devised a distance
ratio parameter, which is defined as the ratio of the “mean between-sample
distance” to the “mean within-sample distance”:
RD??mean between-sample distance
mean within-sample distance?
Bray-Curtis distance, as with other distance indices used to compare two sam-
ples, can be treated as the difference between two normally distributed variates
X ? N(?X, ?2) and Y ? N(?Y, ?2). The distribution of unsigned differences
between these two variates, Dx?y? ? X ? Y?, can be treated as a folded normal
distribution (18) with mean
4?2 ?? ?X?Y?1 ? 2??
where ?X ? Y ? ?X ? ?Yand ??a? ?
0, the “mean within-sample distance” simplifies to ?f?X?
??, and the unsigned distance ratio simplifies to
2 dt. Because ?X ? X ?
?Y ? Y
RD??f?X ? Y?
2? ?1 ? 2??
If?X ? Y
corresponds to an RDvalue of ?1.8. In the case that comparison is made between
samples i and j with two repeats for each sample, the distance ratio for these two
samples could be calculated as
? 2, X and Y have a 5% chance of being the same distribution, which
RD?i,j? ?mean?di1,j1, di1,j2, di2,j1, di1,i2?
If the variation between two sample types is similar to the variation between
repeat sampling events of the same sample, the distance ratio would be close to
1, and thus, the hypothesis that these two samples are different is not supported.
On the other hand, for two samples with a distance ratio of ?2, there is a ?5%
chance that two samples are from the same distribution. Single-factor analysis of
variance was used to determine the P values for each within-sample versus
between-sample comparison. To evaluate intersample differences within subjects
403 and 409, we repeated the library cloning process for each sample type. The
Bray-Curtis dissimilarity indices based on 67 phylotypes (operational taxonomic
units) were used as the distance for this calculation.
Clinical evaluation and sample collection. The eight pre-
menopausal nonpregnant women were asymptomatic for vag-
inal infections, as determined by self-assessment and clinical
examination by the physician (J. S. Weisbaum). The median
age of the women was 26.5 years (range 20 to 45 years), with
seven self-identifying as Caucasian and one as Asian-Indian.
Six vaginal samples were obtained from each subject, providing
a total of 48 samples, from which 16S rRNA gene clone librar-
ies were constructed.
Overall structure and diversity of vaginal bacterial commu-
nities. Approximately 500 clone sequences from the six sam-
ples combined were available for most subjects, while repeat
library construction provided over 1,000 clone sequences each
for subjects 403 and 409. The combined 5,340 16S rRNA gene
clones from all eight subjects were grouped into sequences
representing 67 phylotypes, identified from 67 type strains
from the RDP II database.
Phylogenetic analysis yielded a tree comprising five phyla,
namely Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes,
and Actinobacteria, and 32 genera (Fig. 2). Members of the
phylum Firmicutes (mainly the genus Lactobacillus) were the
most frequently detected clones recovered from the subjects,
accounting for 55% of all clones. Clones with sequences be-
longing to the phyla Proteobacteria and Actinobacteria (mainly
the genera Pseudomonas and Gardnerella, respectively) also
constituted significant portions of the libraries, with each ac-
counting for 15% of all the clones, while those belonging to
Fusobacteria and Bacteroidetes were far less prevalent.
Genus level diversity was variable depending on the phy-
lum, although this may be a reflection of database bias (16).
The phyla Firmicutes and Proteobacteria possessed phylo-
types belonging to 14 and 12 genera, respectively, but the
other three phyla contained phylotypes belonging to only
one to three genera each. At the species level, the genus
Pseudomonas showed the highest diversity, with 19 species,
while Lactobacillus had a total of six species. Five type strain
sequences (Lactobacillus iners, Lactobacillus crispatus, Lac-
tobacillus gasseri, Gardnerella vaginalis, and Pseudomonas
gessardii) closely matched more than 500 clones each, while
22 type strain sequences closely matched only one or two
Variation in microbial composition among subjects. To ex-
amine the overall microbial profile of each individual, a
dendrogram comparing the eight subjects was constructed
based on the Bray-Curtis distances of the phylum-level com-
position of the combined clone libraries for each individual
VOL. 47, 2009HETEROGENEITY OF VAGINAL BACTERIAL COMMUNITIES1183
at UNIVERSITY OF ILLINOIS on June 8, 2009
(Fig. 3). Subjects clustered into four groups with a cutoff
distance of 20%: group 1 (subjects 401 and 402), group 2
(subjects 404, 405, 406, and 408), group 3 (subject 403), and
group 4 (subject 409). The presence and relative abundance
of the 67 phylotypes varied among the groups (Fig. 2 and 3).
Group 1 showed the lowest diversity compared to those of
the other groups, according to Shannon’s and Simpson’s
diversity indices (Table 1), whereas group 4, which had
members from all five phyla represented, exhibited the high-
est diversity. Group 1 was comprised predominantly of Lac-
tobacillus species, constituting approximately 90% of the
total clones sequenced from each individual. Lactobacillus
and Pseudomonas constituted the two most abundant bac-
terial genera found in group 2, with Lactobacillus species
accounting for 60 to 70% of the clones and Pseudomonas
species 20 to 30%. The microbial compositions of groups 3
and 4 differed substantially from those of groups 1 and 2,
with high proportions of sequences closely related to Gard-
nerella vaginalis. In group 3, 46% and 42% of the clones
were closely related to G. vaginalis and Lactobacillus spe-
FIG. 2. Phylogenetic distribution of vaginal bacteria from eight healthy, asymptomatic subjects. Numbers of clones for each phylotype are
indicated in parentheses. The tree was produced using DNADIST and FITCH in PHYLIP. Bootstrap values of ?50 from 100 resamplings
(? indicates bootstrap value of ?50, and ? represents no clustering in the method) are indicated at each node (Fitch/parsimony/maximum
likelihood). The scale bar represents 0.02 substitutions per site. Thermotoga maritima was used as the outgroup. The right side of the figure displays
relative abundances of the phylotypes by subject and by group. The numbers above the abundance columns for the subjects correspond to the
subject identification number. G1 to G4 correspond to the four groups into which the subjects were divided.
1184 KIM ET AL.J. CLIN. MICROBIOL.
at UNIVERSITY OF ILLINOIS on June 8, 2009
cies, respectively. In group 4, G. vaginalis represented 27%
of the clones, followed by clones related to Pseudomonas
species (21%) and Leptotrichia amnionii (16%).
Nugent scoring of phylogenetic groups. Nugent scoring of
Gram-stained smears was performed to compare the microbial
profiles determined by 16S rRNA gene clone library analysis to
the vaginal bacteria detected microscopically in each subject.
Examination of the microbiota by Gram staining revealed that
the six subjects in groups 1 and 2 had normal vaginal micro-
biota (Nugent score ? 0), while the subject in group 3 (no. 403)
had intermediate or altered vaginal microbiota (Nugent score ?
6) and the subject in group 4 (no. 409) had a Nugent score of
9, indicative of vaginal microbiota consistent with BV, despite
self-reporting as asymptomatic and having no overt clinical
Variation in microbial composition by sample type within
subjects. The influence of anatomical site and sampling
method on the composition of bacterial communities within
each individual is reflected by the heterogeneity of the popu-
lation distribution patterns among samples within individuals
(Fig. 4). Clustering based on the microbial composition of the
vaginal samples within each individual revealed that grouping
of samples varied from subject to subject, indicative of the
heterogeneity of samples both between and within individuals.
Lactobacillus species were the most abundant bacteria in all
the sample types in subjects 401 and 402. In subjects 404, 405,
406, and 408, Lactobacillus and Pseudomonas species were
dominant in all the samples, but the relative abundances of
each varied significantly among the sample types within and
between individuals. G. vaginalis was abundant in all the sam-
ples from subject 403, but again, the relative abundances varied
among the sample types. Substantial variation in bacterial com-
position was observed among all of the samples in subject 409,
with none of the samples showing similarity to each other in
their bacterial compositions.
Diversity of microbiota within and among different sample
types. Clustering of subjects varied considerably depending on
the sample type (see Fig. S1 in the supplemental material). We
performed pairwise comparisons among the 48 sample types
from all eight subjects using ?-LIBSHUFF analysis (24), which
tests the differences among bacterial 16S rRNA gene libraries.
The results revealed the bacterial content of some samples
within the same subject to be significantly (P ? 0.001) different
from other samples, while a number of samples were found to
be subsets of each other (see Table S1 in the supplemental
material). Libraries from the lavage samples of most of the
subjects were subsets of the other libraries from the same
subject. Compared to the other samples, the lavage samples
detected the least diversity in the vaginal microbiota and ex-
hibited a complete absence of Proteobacteria sequences (e.g.,
Pseudomonas) (see Fig. S2 in the supplemental material). The
lavage samples were also the only samples that had no unique
sequences that were not also present in one of the other sam-
ple types, and removing the lavage library from the combined
libraries of each subject did not affect the types of bacteria
detected for that individual (see Fig. S3 in the supplemental
material). As swabs are the most commonly collected vaginal
samples in published studies, the bacterial diversity detected by
the combined swab samples alone was also examined. In most
subjects, fewer bacterial types were detected using the three
swab samples alone than those seen in the clone libraries of all
six samples combined for each individual (see Fig. S3 in the
supplemental material). The scrape samples showed the great-
est bacterial diversity. Indeed, the two scrape samples alone
detected as much diversity as all six vaginal samples combined
(see Fig. S3 in the supplemental material).
Distance relationships of microbial profiles from different
sample types. Bray-Curtis distance analysis, which addresses
the differences in population profiles based on specified clas-
sification criteria (i.e., binning by closely related representa-
tives) rather than strictly considering phylogenetic sequence
similarity, was performed for all sample types within each sub-
ject (see Table S2 in the supplemental material). When the
distances were calculated at three different levels (67 phylo-
types, 32 genera, and five phyla), the intersample variation
could be identified clearly from the distance values in each
case. However, consistent patterns in the sample-related vari-
ation of the bacterial populations were not observed among
the eight subjects. In some subjects, only one sample (e.g., the
FIG. 3. Grouping of subjects by bacterial composition at the phy-
lum level. The histogram (lower panel) shows the phylum level bacte-
rial composition of each subject, and the dendrogram (upper panel)
displays the clustering of the eight subjects. Four groups were identi-
fied using a cutoff distance value of 20%. Over 500 clone sequences
each were analyzed for groups 1 and 2, while about 1,000 clone se-
quences each were analyzed for groups 3 and 4.
TABLE 1. Bacterial diversity of vaginal microbiota by subjects
Value for indicated subjects in group:
401402408 404405 406 403 409
VOL. 47, 2009HETEROGENEITY OF VAGINAL BACTERIAL COMMUNITIES 1185
at UNIVERSITY OF ILLINOIS on June 8, 2009
lower scrape sample for subject 401) appeared different from
all the other samples within that individual. In other subjects,
the six samples clearly fell into separate groups, such as those
of subject 405, where the lower scrape and upper scrape sam-
ples were distinguishable from the swab and lavage samples.
And yet, all of the samples for subject 409 appeared to be quite
heterogeneous with respect to each other.
Evaluation of potential cloning bias. To test the possibility
that the observed heterogeneity of the distribution patterns for
the microbial populations might be due to cloning bias, we
repeated the construction and sequencing of the libraries for
two subjects. Subjects 403 and 409, whose samples showed the
most microbial diversity and differed substantially from those
of the other subjects in the composition of their vaginal bac-
teria, were selected for the construction of repeat libraries to
confirm the presence of the highly diverse microbiota observed
in these individuals. No differences were found in the bacterial
compositions of the original and repeat clone libraries by using
?-LIBSHUFF analysis (data not shown). Bray-Curtis dissimi-
larity distances for the repeat libraries were also used to cal-
culate distance ratios of between-sample versus within-sample
distances, which is equivalent to the statistical distance, as
detailed in Materials and Methods. This allowed us to deter-
mine whether the distance between two different samples
within the same subject was greater than the distance between
repeat libraries of the same sample from that individual (Fig.
5). The ratios for the sample types within each of these subjects
(Fig. 5A), most of which were ?1, confirmed that the variation
found among sampling sites or sampling techniques was not
the result of variation that would be expected from repeat
library cloning of the same sample. The distance ratios ob-
tained correlated well with the Bray-Curtis distances from the
combined libraries of the repeats (Fig. 5B). Using this distance
ratio index, after considering the sampling variation for the
FIG. 4. Clusteringofvaginalsampleswithinindividualsbasedonmicrobialcomposition.Thehistograms(lowerportionofeachpanel)showthephylumlevel
bacterial composition of each subject, and the dendrograms (upper portion of each panel) display the clustering of the sample types within each of the eight
subjects. CX, swab cervix library; FX, swab fornix library; OT, swab outer library; SL, scrape lower library; SU, scrape upper library; LA, lavage library.
FIG. 5. Distance ratios of between-sample versus within-sample
distances for repeat libraries from subjects 403 and 409. (A) Bray-
Curtis dissimilarity distances for the repeat libraries for the two most
diverse subjects (no. 403 and 409) used to calculate distance ratios. The
P values for single factor analysis of variance comparing between-
sample distances versus within-sample distances are denoted as fol-
lows: ?, P ? 0.05; ??, P ? 0.01; and ???, P ? 0.001. (B) Linear
correlation of Bray-Curtis distances with the calculated distance ratios.
1186 KIM ET AL.J. CLIN. MICROBIOL.
at UNIVERSITY OF ILLINOIS on June 8, 2009
repeat libraries from subject 403, the cervix and fornix swab
samples were not different from each other (distance ratio of
?1). Likewise, the cervix swab and lavage samples were not
different from each other (distance ratio of ?1). However, the
fornix swab and outer swab samples were different from each
other (distance ratio of 4.6). For subject 409, most of the
samples were different from each other (distance ratios of 1.8
Normal as well as abnormal microbiotas play a critical role
in the progression and outcome of complex microbial diseases
(e.g., see references 19, 28, 32, and 36). In order to better
understand the disease process, to gain clearer understanding
of the influence of the microbiota that are present, and to
better evaluate methods for diagnosing and treating these
complex diseases, it is important to understand the impact that
variability in collection protocols and sampling techniques may
have on diagnosis and data interpretation.
Here, we report considerable variation between the micro-
biotas among samples collected from three vaginal sites (cer-
vix, fornix, outer vaginal canal) and by three different sampling
methods (lavaging, swabbing, scraping). Previous culture-
based studies have reported differences in site-specific vaginal
microbial profiles (2, 3, 21), but the scope of these studies was
limited due to cultivation biases. This is the first study exam-
ining niche variation in the vaginal tract by using molecular
methods, which allowed for greater resolution in the detection
of site-specific differences. In addition, this is the first study to
address potential sampling bias introduced by use of swabs
only, which may not adequately collect adherent bacteria. Fi-
nally, results for culture-independent analysis of the influence
of sampling methods on the detection of vaginal microbes have
also not been previously reported.
The lavage sample detected the least diversity in the vaginal
microbiota and may not be representative of the true diversity
of the vaginal bacteria. Though the lavage sample was the most
dilute of the six samples collected, dilution would not be ex-
pected to alter the overall microbial composition of the sam-
ples, merely the total quantity of microbes within that sample.
An interesting observation was the complete lack of Proteobac-
teria sequences from the lavage samples of all the subjects,
even though Proteobacteria constituted the second largest rep-
resentation in the overall vaginal clone library. Little or no
Fusobacteria, Actinobacteria, or Bacteroidetes were found in the
three swab or lavage clone libraries for six of the eight subjects
(groups 1 and 2), even though Fusobacteria, Actinobacteria,
and, to a lesser extent, Bacteroidetes were present in the scrape
samples, which presumably would facilitate the collection of
more adherent organisms. Prior studies have shown that the
vaginal microbiota includes organisms that form loosely and
tightly tissue-adherent biofilms (9, 29), possibly accounting for
the differences in the bacteria detected among the three sam-
pling methods utilized. G. vaginalis, a member of the phylum
Actinobacteria, forms adherent biofilms (29), and this may ex-
plain the greater prevalence of Actinobacteria in the scrape
samples of some subjects. These results suggest that a single
sample from an individual might not be sufficient to reflect the
complexity of the vaginal microbiota within a subject, and
these results provide a framework for microbial ecologists and
population biologists who seek to identify and characterize
factors that underlie the establishment and maintenance of
barriers that separate and define microenvironments.
In this study, we were particularly concerned about causing
perturbations in the microbial community composition at sites
that were swabbed or scraped and, thus, collected a single
sample from each site by any particular method. Nonetheless,
we collected both a swab sample and a scrape sample from the
lower one-third region of the vaginal canal of each individual.
For six of the eight subjects, the microbial community compo-
sitions of the swab and scrape samples were considerably dif-
ferent. It is not currently clear whether these differences were
strictly due to differences in the sampling methods or due to
perturbations at the sampling site during collection, but this
work underscores the importance of considering the potential
effects of sample collection methodology on microbial commu-
The composition of the vaginal microbiota varied substan-
tially among the subjects in this study, in agreement with
observations from other culture-independent studies of the
vaginal microbiota (14, 17, 31, 33, 35, 36). Individuals from
group 1 and group 2 all had Nugent scores of 0, consistent with
what is generally considered a “healthy” vaginal microbiota
(22). The vaginal microbiotas of group 1 subjects exhibited the
least microbial diversity, and all the sample types were domi-
nated by Lactobacillus spp. and, thus, most closely resembled
what is conventionally considered to be a “healthy” microbial
composition (1, 8, 10). Lactobacillus and Pseudomonas species
dominated all the samples from group 2, but the relative abun-
dances of each varied widely among the sample types within
and between individuals. Given the abundance of Pseudomo-
nas in most of the subjects in this study and in those from a
recent study by Hyman et al. (17), Pseudomonas spp. may be
previously unrecognized common members of the healthy vag-
Gardnerella vaginalis was present in all the samples from
subject 403 (group 3) and in most samples from subject 409
(group 4), but the relative abundances varied among the sam-
ple types. The presence of G. vaginalis sequences in the clone
libraries of these two subjects is consistent with the Gram
staining results for these individuals. While all of the subjects
in this study were asymptomatic and did not exhibit any clinical
signs of BV, the Nugent scores and the greater microbial di-
versities observed for subjects 403 and 409 suggest that subject
403 may have been transitioning into or out of disease and
subject 409 may have had asymptomatic BV at the time of
specimen collection. Alternatively, it is possible that our un-
derstanding of BV is incomplete, and a high Nugent score does
not necessarily reflect a diseased state in all cases. Moreover,
subject 409, who exhibited the greatest heterogeneity in bac-
terial composition among all of the sample types, reported
having been infected with chlamydia in the 6 months prior to
the study, indicating that the recently perturbed microbiota
may have not yet recovered from the infection. However, this
subject tested negative for chlamydia at the time of sample
collection, and Chlamydia 16S rRNA gene sequences were not
detected in the clone libraries from this individual. Interest-
ingly, subjects 403 and 409 were also the only two subjects
reporting new sexual partners within the 6 months prior to
VOL. 47, 2009HETEROGENEITY OF VAGINAL BACTERIAL COMMUNITIES 1187
at UNIVERSITY OF ILLINOIS on June 8, 2009
sample collection. History of multiple sexual partners is a
known risk factor for BV (4, 26, 27), but given the small sample
size, it is not possible to draw any conclusions regarding sexual
history and vaginal microbiota in these study participants.
These results are highly significant for medical microbiologists
who seek to correlate microbial community composition to the
pathophysiology of vaginal diseases, and especially for clinical
practitioners interested in predictive measures for vaginal health
and disease, susceptibility to sexually transmitted disease, repro-
ductive fecundity, and pregnancy outcome. Notably, standard
clinical practice today mandates a single swab of the vaginal area,
but protocols are poorly defined. The data presented here indi-
cate that the bacterial diversity detected by combining swab sam-
ples only from three different locations in the vaginal canal is
lower than that detected using both swab and scrape samples
from multiple locations within an individual. The broader impli-
cation of this demonstration of the differences between samples
within individuals is that it highlights the importance of the sam-
vaginal microbial communities within individuals.
The methods utilized in this study are applicable to both
clinical and laboratory settings. Our clinical partners have been
implementing the specimen collection methods described here
with relative ease. Clinical laboratory techniques are changing
rapidly and exploring the use of PCR and DNA gene-chip
technologies for diagnostics. Consequently, it is very likely that
culture-independent bacterial detection techniques such as 16S
sequencing could be utilized in a clinical laboratory setting in
the near future.
These findings could significantly impact specimen collec-
tion protocols and sampling techniques for experimental stud-
ies and for clinical diagnostic evaluation and treatment of
vaginal disease, as microbes indicative of an unhealthy vaginal
state may be absent or underrepresented in one sampling site
or by one sampling method but may be predominant in an-
other. This niche variation is particularly important with regard
to obstetric and neonatal care in considering the appropriate
collection technique for the determination of group B strep-
tococcal colonization in pregnant women, a critical risk factor
for adverse postpartum outcome (15, 23). Thus, based on the
results of this study, an overall scoop of the entire vaginal tract
that includes collection of adherent bacteria might provide a
more representative view of the vaginal microbial composition
and may be important for accurate clinical evaluation of indi-
viduals, especially those with highly heterogeneous microbiota
or those who may be transitioning into disease.
This work was supported in part by the Research Board and the
Institute for Genomic Biology of the University of Illinois at Urbana-
Champaign and the Carle Foundation Hospital.
We thank Michelle Hughes, Barbara Hall, Cindy Fraser, and Ann
Benefiel for their clinical and administrative assistance with sample
collection. We thank Brian Ho, Rachel Whitaker, Angela Kent, and
Abigail Salyers for helpful discussions.
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