# Temporal Shifts in Microbial Communities in Nonpregnant African-American Women with and without Bacterial Vaginosis

Authors: John Wertz; Natasha Isaacs-Cosgrove; Claudia Holzman; Terence L. Marsh
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2008)

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## Abstract

Bacterial vaginosis (BV) has been described as an increase in the number of anaerobic and facultatively anaerobic bacteria relative to lactobacilli in the vaginal tract. Several undesirable consequences of this community shift can include irritation, white discharge, an elevated pH, and increased susceptibility to sexually transmitted infections. While the etiology of the condition remains ill defined, BV has been associated with adverse reproductive and pregnancy outcomes. In order to describe the structure of vaginal communities over time we determined the phylogenetic composition of vaginal communities from seven women sampled at multiple points using 16S rRNA gene sequencing. We found that women with no evidence of BV had communities dominated by lactobacilli that appeared stable over our sampling periods while those with BV had greater diversity and decreased stability overtime. In addition, onlyLactobacillus iners was found in BV positive communities.


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## Body

## 1. Introduction

The relationship between biodiversity and ecosystem stability has been
critically discussed  and investigated over the past decade
[1–10]. Most reports have provided evidence
suggesting that greater biodiversity leads to greater system stability in the
face of stress. A number of theoretical constructs have been created to account
for the relationship between biodiversity and stability [3, 11, 12] in which high biodiversity
within an ecosystem is frequently equated with a level of functional
redundancy. Thus in periods of stress, the loss of a species is not
catastrophic given a level of redundancy.Microbial communities provide a remarkable system for investigating these
relationships. Many apparently stable microbial communities are constructed of
hundreds or thousands of species. Notable examples are the human intestinal
microflora with an estimated 500–600 species [13, 14] and soil with an estimated
2000–3000 species/gram
[15]. Perturbations resulting in
significant community shifts have been detected in both of these communities (e.g.,
[16]), but their stability has not
been carefully measured nor has the level of biodiversity been robustly
correlated with stability. Interestingly, Fernández et al. [17] described a bioreactor with
functional stability but apparent  dynamicism in the phylogenetic composition of
the community throughout the experiment. This is consistent with a level of
functional redundancy among the species present that maintained the overall
process in spite of phylogenetic shifts within the community.From the perspective of the biodiversity-stability debate, the vaginal
tract is an interesting ecosystem. In a large percentage of females the vaginal
microbial community is relatively simple and dominated by one or several
species ofLactobacillus [18–23]. However, when this simple
community is replaced by bacterial vaginosis (BV), the shift is from the near
monoculture of lactobacillus to a community with orders of magnitude greater
phylogenetic diversity, especially in regards to Gram-positive anaerobes [19, 24–27]. Only a few investigators
have addressed the stability of the community over time for either BV negative
or BV positive females (e.g., [28, 29]).In the work described herein, we present phylogenetic assessments of the
vaginal microbial community from nonpregnant women. Multiple samples were taken
from each woman on a monthly schedule, and the phylogenetic composition of the
communities was determined by comparative sequence analysis of 16S rRNA gene
libraries. Our goal was to compare the microbial community structure in BV
positive and BV negative women over time and examine whether diversity
correlated with greater stability.


## 2. Methods

### 2.1. Study Sample

Vaginal
samples used in this study were collected as part of a small, randomized
clinical trial (RCT) of vaginal douching cessation. The primary goals of the
RCT were to assess the acceptability of douching cessation and an at home data
collection protocol over a four-month period. Secondary goals included
describing BV presence/absence throughout the study period, and identifying
factors associated with BV (e.g., phase of menstrual cycle, lifestyle). The
study was conducted on a college campus and eligibility criteria included
douching currently at least once per month and not being pregnant. Women were
enrolled over a six-week period and total sample size was limited to the first
45 eligible women. At enrollment, women met with study personnel at the campus
clinic to review and sign consent forms and to complete a baseline
questionnaire.  Participants were then
randomized either to continue usual douching patterns or to refrain from all
vaginal douching. At enrollment, women self-collected two swabs for baseline
data on vaginal microflora. Thereafter, study participants were asked to
complete a daily diary and self-collect three vaginal swabs a week (one on the
weekend and two spread across the weekdays) for four months. Diaries included
information about days of menses, sexual activity, contraceptive use, vaginal
douching, vaginal symptoms, bathing, showering, illness, medications, and
stress level. Diary sheets and slides were returned by mail weekly.
Participants also returned to the campus clinic every two weeks at which time a
swab for BV assessment was collected and vaginal pH was measured. Once per
month an additional swab was collected, placed in sterile saline and frozen at
−80°C.For the current study, frozen vaginal
fluid samples from seven women enrolled in the RCT were selected for further
study. All seven women were African-American, and they had been assigned to the
intervention arm (i.e., asked to refrain from vaginal douching) and reported
that they did not use any form of hormone-based contraceptive.  For comparison purposes, five of the seven
women were selected because, throughout the four months of the RCT, all of
their vaginal smears were negative for BV; the other two women were selected
because they frequently showed evidence of BV during the same study period.
Clone libraries of 16S rRNA genes were constructed from 2-3 monthly vaginal
samples of each BV negative woman and 4 monthly samples from BV positive women.


### 2.2. Nugent Scoring for BV

A single
microbiologist with training in the Nugent method for scoring BV [30] evaluated all vaginal smears
while blinded to the randomization assignment and data from the diaries. In a
previous study with the same microbiologist and a second microbiologist, the
kappa for BV positive (Nugent score ≥7) versus BV negative smears
was .81 [31]. Nugent scores range from 0
to 10, the higher scores are indicative of more Gram-negative aerobes
and Gram-positive anaerobes and fewer lactobacilli. A Nugent score of 0–3 is considered
BV negative, 4–6 is
intermediate, and 7–10 is BV
positive.


### 2.3. Extraction of DNA and PCR Amplification

Microbial DNA was extracted using MoBio
Soil DNA extraction kits as follows. Frozen vaginal swabs were soaked in 70%
ethanol overnight. The tip was removed from the tube and residual ethanol was
squeezed out on the side of the tube. The swab tip was then cut off and placed
into the MoBio extraction tube and stored at −20°C until extraction. The
ethanol wash was centrifuged for 30 minutes at 10,000 × g in a microfuge and
the resulting pellet was resuspended in 200 μL of water and transferred to the
MoBio extraction tube with the swab tip. The combined pellet and swab tip were
lysed by bead beating for 1.5 minutes and then extracted according to the
manufacturer’s instructions. This protocol ensured that both free DNA derived
from lysed cells and DNA from intact cells were collected from the samples.
Pilot PCR reactions of 25 μL were performed using bacterial domain specific
primers 27F (5′-AGA GTT TGA TCM
TGG CTC AG-3′) and 1389R
(5′-AGC GGC GGT GTG TAC AAG-3′) [32]. The PCR reaction volume was
25 μL with 30 ng template DNA. Reactions contained 1X buffer (Invitrogen), 1.5 mM MgCl2,
0.25 mM of dNTPs, and 0.2 μM of each primer and 0.6 units of Taq polymerase (Invitrogen). Cycling was initiated with an initial
denaturation of 3 minutes at 95°C followed by 25 cycles of 45 seconds at 95°C,
45 seconds at 56°C and 1 minute at 72°C, followed by a 5-minute extension at
72°C. PCR products were analyzed on agarose gels stained with ethidium bromide.
Reactions with the appropriate size PCR product were cloned using Invitrogen’s TOPO cloning kit. Putative clones with
inserts were picked, screened and sequenced at the technology center at MSU. Sequences
were deposited at GenBank (EF364727
to EF365525 (low Nugent scores) and EF365526 to EF366669 (high Nugent scores)).


### 2.4. Phylogenetic and Statistical Analyses

Each 16S rRNA gene clone was assigned a
preliminary phylogenetic affiliation by sequence comparison to the Ribosomal
Database Project II using the sequence match tool [33]. Sequences were checked for
chimerae using the Chimera Check program [34], and sequences shorter than
550 nucleotides were removed. Sequences that were not clearly assigned at the
genus level were compared to the Genbank nucleotide database using BLAST [35].  Sequences were aligned based on secondary
structure to the 16S rRNA gene sequence database ssuJan03 in the ARB software
package (http://www.arb-home.de/) using
the Fast Aligner tool [36]. Unaligned or ambiguously
aligned nucleotides were corrected manually. For all subsequent analyses, 503
unambiguously aligned nucleotides corresponding to positions 119 to 638 in Escherichia coli were used.For
phylogenetic analyses, when closely related sequences were not identified in
the ARB database, relatives were found by a BLAST search of the Genbank
database and incorporated into ARB. Phylogenetic trees were constructed using
the neighbor-joining method with a Felsenstein correction. A minimum
evolutionary distance method in PAUP* was used for bootstrap analysis of the
same data.Differences
in the libraries were tested by pairwise comparison of PHYLIP-formatted
distance matrices for each library using webLIBSHUFF version 0.96 [37], which combines preLIBSHUFF [38] and LIBSHUFF version 1.22 [39]. For further community
analyses, the sequences were grouped into operational taxonomic units (OTUs)
using DOTUR [39]. A distance of 3% was used to
define an OTU, and is hereafter denoted as OTU0.03. A 3% dissimilarity
in 16S rRNA gene sequences is typically, though controversially, thought to
represent a species-level delineation [40]. For each participant, the
two-, three-, or four-clone libraries were combined and the Chao1 richness and
Simpson diversity (D) estimators were calculated as implemented in the DOTUR
program. The Simpson index of diversity was calculated as 1D. The Chao1
estimator, at an OTU0.03 cutoff, can be thought to represent the
estimated number of species in an environment. The Simpson index of diversity
is an estimate that takes into account the richness as well as the evenness
(number of each species). To obtain a quantitative measure of the OTU0.03 similarity between libraries sampled from the same participant, the Yue and Clayton nonparametric maximum likelihood method
was calculated using the SONS software [41].


## 2.1. Study Sample

Vaginal
samples used in this study were collected as part of a small, randomized
clinical trial (RCT) of vaginal douching cessation. The primary goals of the
RCT were to assess the acceptability of douching cessation and an at home data
collection protocol over a four-month period. Secondary goals included
describing BV presence/absence throughout the study period, and identifying
factors associated with BV (e.g., phase of menstrual cycle, lifestyle). The
study was conducted on a college campus and eligibility criteria included
douching currently at least once per month and not being pregnant. Women were
enrolled over a six-week period and total sample size was limited to the first
45 eligible women. At enrollment, women met with study personnel at the campus
clinic to review and sign consent forms and to complete a baseline
questionnaire.  Participants were then
randomized either to continue usual douching patterns or to refrain from all
vaginal douching. At enrollment, women self-collected two swabs for baseline
data on vaginal microflora. Thereafter, study participants were asked to
complete a daily diary and self-collect three vaginal swabs a week (one on the
weekend and two spread across the weekdays) for four months. Diaries included
information about days of menses, sexual activity, contraceptive use, vaginal
douching, vaginal symptoms, bathing, showering, illness, medications, and
stress level. Diary sheets and slides were returned by mail weekly.
Participants also returned to the campus clinic every two weeks at which time a
swab for BV assessment was collected and vaginal pH was measured. Once per
month an additional swab was collected, placed in sterile saline and frozen at
−80°C.For the current study, frozen vaginal
fluid samples from seven women enrolled in the RCT were selected for further
study. All seven women were African-American, and they had been assigned to the
intervention arm (i.e., asked to refrain from vaginal douching) and reported
that they did not use any form of hormone-based contraceptive.  For comparison purposes, five of the seven
women were selected because, throughout the four months of the RCT, all of
their vaginal smears were negative for BV; the other two women were selected
because they frequently showed evidence of BV during the same study period.
Clone libraries of 16S rRNA genes were constructed from 2-3 monthly vaginal
samples of each BV negative woman and 4 monthly samples from BV positive women.


## 2.2. Nugent Scoring for BV

A single
microbiologist with training in the Nugent method for scoring BV [30] evaluated all vaginal smears
while blinded to the randomization assignment and data from the diaries. In a
previous study with the same microbiologist and a second microbiologist, the
kappa for BV positive (Nugent score ≥7) versus BV negative smears
was .81 [31]. Nugent scores range from 0
to 10, the higher scores are indicative of more Gram-negative aerobes
and Gram-positive anaerobes and fewer lactobacilli. A Nugent score of 0–3 is considered
BV negative, 4–6 is
intermediate, and 7–10 is BV
positive.


## 2.3. Extraction of DNA and PCR Amplification

Microbial DNA was extracted using MoBio
Soil DNA extraction kits as follows. Frozen vaginal swabs were soaked in 70%
ethanol overnight. The tip was removed from the tube and residual ethanol was
squeezed out on the side of the tube. The swab tip was then cut off and placed
into the MoBio extraction tube and stored at −20°C until extraction. The
ethanol wash was centrifuged for 30 minutes at 10,000 × g in a microfuge and
the resulting pellet was resuspended in 200 μL of water and transferred to the
MoBio extraction tube with the swab tip. The combined pellet and swab tip were
lysed by bead beating for 1.5 minutes and then extracted according to the
manufacturer’s instructions. This protocol ensured that both free DNA derived
from lysed cells and DNA from intact cells were collected from the samples.
Pilot PCR reactions of 25 μL were performed using bacterial domain specific
primers 27F (5′-AGA GTT TGA TCM
TGG CTC AG-3′) and 1389R
(5′-AGC GGC GGT GTG TAC AAG-3′) [32]. The PCR reaction volume was
25 μL with 30 ng template DNA. Reactions contained 1X buffer (Invitrogen), 1.5 mM MgCl2,
0.25 mM of dNTPs, and 0.2 μM of each primer and 0.6 units of Taq polymerase (Invitrogen). Cycling was initiated with an initial
denaturation of 3 minutes at 95°C followed by 25 cycles of 45 seconds at 95°C,
45 seconds at 56°C and 1 minute at 72°C, followed by a 5-minute extension at
72°C. PCR products were analyzed on agarose gels stained with ethidium bromide.
Reactions with the appropriate size PCR product were cloned using Invitrogen’s TOPO cloning kit. Putative clones with
inserts were picked, screened and sequenced at the technology center at MSU. Sequences
were deposited at GenBank (EF364727
to EF365525 (low Nugent scores) and EF365526 to EF366669 (high Nugent scores)).


## 2.4. Phylogenetic and Statistical Analyses

Each 16S rRNA gene clone was assigned a
preliminary phylogenetic affiliation by sequence comparison to the Ribosomal
Database Project II using the sequence match tool [33]. Sequences were checked for
chimerae using the Chimera Check program [34], and sequences shorter than
550 nucleotides were removed. Sequences that were not clearly assigned at the
genus level were compared to the Genbank nucleotide database using BLAST [35].  Sequences were aligned based on secondary
structure to the 16S rRNA gene sequence database ssuJan03 in the ARB software
package (http://www.arb-home.de/) using
the Fast Aligner tool [36]. Unaligned or ambiguously
aligned nucleotides were corrected manually. For all subsequent analyses, 503
unambiguously aligned nucleotides corresponding to positions 119 to 638 in Escherichia coli were used.For
phylogenetic analyses, when closely related sequences were not identified in
the ARB database, relatives were found by a BLAST search of the Genbank
database and incorporated into ARB. Phylogenetic trees were constructed using
the neighbor-joining method with a Felsenstein correction. A minimum
evolutionary distance method in PAUP* was used for bootstrap analysis of the
same data.Differences
in the libraries were tested by pairwise comparison of PHYLIP-formatted
distance matrices for each library using webLIBSHUFF version 0.96 [37], which combines preLIBSHUFF [38] and LIBSHUFF version 1.22 [39]. For further community
analyses, the sequences were grouped into operational taxonomic units (OTUs)
using DOTUR [39]. A distance of 3% was used to
define an OTU, and is hereafter denoted as OTU0.03. A 3% dissimilarity
in 16S rRNA gene sequences is typically, though controversially, thought to
represent a species-level delineation [40]. For each participant, the
two-, three-, or four-clone libraries were combined and the Chao1 richness and
Simpson diversity (D) estimators were calculated as implemented in the DOTUR
program. The Simpson index of diversity was calculated as 1D. The Chao1
estimator, at an OTU0.03 cutoff, can be thought to represent the
estimated number of species in an environment. The Simpson index of diversity
is an estimate that takes into account the richness as well as the evenness
(number of each species). To obtain a quantitative measure of the OTU0.03 similarity between libraries sampled from the same participant, the Yue and Clayton nonparametric maximum likelihood method
was calculated using the SONS software [41].


## 3. Results

In the seven women selected for this study, vaginal pH ranged from 4.0 to
5.8 and, as expected, pH was highest when BV was present (Table1).  For most women, samples were obtained at
different menstrual weeks. Among BV positive women, there were no reports of
antibiotic or antifungal use, and intercourse was infrequent in the week before
vaginal sampling.Table 1
Relevant clinical and 16S
rRNA gene clone library information for the seven participants in this study.
ParticipantLibraryBV ScorepHMenstrual cycle (week)Frequency of intercourse (week prior)Antibiotic/ antifungal (previous month)No. of  clones in libraryPhylogenetic affiliation of dominant phylotypeDominant phylotype (% of library)11a04.420No85Lactobacillus
crispatus981b14.430No86L. crispatus9822a04.040Antibiotic50L. crispatus1002b04.040No91L. crispatus9933a24.730Antibiotic53Lactobacillus gasseri623b44.730No58L. gasseri9544a14.444Antifungal79Lactobacillus iners994b24.740Antifungal50L. iners984c04.46#1Antifungal74L. iners9955a04.043No53L. iners915b44.724No60L. iners985c0*5#0No60L. crispatus9366a85.020No147L. iners596b85.841No170L. iners416c85.830No162Leptotrichia amnionii366d44.05#0No159L. iners7677a85.030No119Megasphaera sp.627b95.030No165L. iners547c85.521No130L. iners357d85.510No92Prevotella buccalis40*Missing data.
Long# menstrual cycle.We evaluated the structure of the microbial communities from the seven
women described in Table1 with 16S rRNA gene  libraries. A total of 20 libraries  were made from both low- (5 women and 12 libraries) and high-(2 women and 8
libraries) Nugent scoring women. A total of 1,943 sequences were analyzed with
library sizes raging from 50 to 170 clones (Table 1). Lactobacillus was the numerically dominant genus in 17 of the
libraries. Three of the libraries from high-Nugent scoring women were dominated
by Leptotrichia/Sneathia, Prevotella, and Megasphaera, respectively. In
total, 28 genera were detected within the Firmicutes, Bacteriodetes,
Actinobacteria, Proteobacteria, and Fusobacteria phyla. In the BV positive women,
20 different genera were detected while only 14 (of which 9 were singletons)
were identified in the BV negative women.Figure1 presents the relative abundance of the detected genera in all of
the libraries. The top 12-community composition profiles represent the communities
from the five individual women with low-Nugent scores. These communities were
dominated by lactobacilli which usually constituted 91% of the community or
greater. The exception to this was library 3a where Lactobacillus constituted only 62% of the clones.Figure 1
Pie chart representations of the vaginal microbial
community structure between participants (top to bottom) and within each
participant, over time (left to right) as inferred by 16S rRNA gene libraries.
The percentage of each library consisting of clones related to members of theLactobacillus genus is given.The eight libraries derived from two women with high-Nugent scores are
presented in the bottom two rows of community composition profiles in Figure1.
These revealed considerably more phylogenetic diversity than that found in
low-Nugent scoring communities, consistent with the morphological basis of the
Nugent scoring system and previously recorded observations [30]. Ten genera were identified in
these libraries that were not detected in libraries from low-Nugent scoring
women. Most of these genera displayed considerable volatility over time. For
example, in woman #6, the genus Megasphaera constituted 22%, 3%, 27%, and 10% of libraries A, B, C, and D,
respectively. This irregular flux in clone numbers was also seen in Prevotella in woman number 7.  Moreover, the lactobacilli were also volatile
in clone numbers over time and were, in general, greatly reduced in numbers in
women with high-Nugent scores. This is in contrast to libraries from low-Nugent
scoring samples where lactobacilli were routinely high and constant in clone
numbers over time.To quantitate these diversity differences we applied a suite of
ecological and statistical measurements to these libraries (Table2).  The Simpson’s diversity index revealed at
least a twofold difference between low and high-Nugent scoring communities
while the Chao species richness similarly revealed substantial differences
between these two groups.  The Yue and Clayton
analysis [41] measures library
similarities. In this table, we calculate intra-woman library similarities and
then compare these across the range of Nugent scores. All libraries with low-
Nugent scores had high similarity (>79%) whereas the high-Nugent scoring
libraries had low similarities (<44%).
On visual inspection of these libraries, it was clear that there was
structural instability in the community over time. Nugent scores did not reveal
subtleties of phylogenetic composition as demonstrated by comparing community
profiles 3a and 6d.Table 2
Relationship between BV
score and the diversity, richness, and stability of the vaginal microbial community.
Participant1BV score2Simpson diversity (1D)Chao1 species richnessLibrary similarity (%)31 (2)0.50.15797.0±1.92 (2)00.034100.0±0.13 (2)30.381479.1±8.64 (3)10.037100±0.15 (3)1.30.331591.0±5.06 (4)70.752743.1±4.67 (4)8.30.852238.2±6.91Numbers in parentheses represent the total number of
clone libraries for that participant.
2Mean of BV scores given in
Table 1.
3Calculated by the nonparametric maximum likelihood estimator of Yue and Clayton. Values ± SE.Analysis of the microbial communities among and between participants with
high- and low-Nugent scores showed that approximately 95.0% (758 out of 799) of
the clones from the low-Nugent scoring women were lactobacilli (Table3). Of
the remaining 5%, most were identified as streptococci (19 clones) or
pseudomonads (9 clones).  The genus Lactobacillus also contained the most
number of clones of any other genus identified in the participants with high-Nugent
scores, though the lactobacilli only accounted for 38.3% (438 out of 1144) of
the total. A majority of the remaining clones grouped with the genera Prevotella (17.3%), Megasphaera (15.7%), Atopobium (7.5%), Sneathia (7.3%), Dialister (3.6%), and Cryptobacterium (2.4%). Of these, only Sneathia was not consistently present in
all eight libraries (Figure 1). No clones belonging to any of these genera were
obtained from participants with low-Nugent scores.Table 3
Phylogenetic affiliation of 16S rRNA gene clones obtained from participants with and without bacterial vaginosis.
PhylumGenus1Participants with BVParticipants without BVTotalFirmicutesLactobacillus4387581196Megasphaera1800180Dialister41041Streptococcus31922Acetivibrio15015Aerococcus16016Micromonas808Gemella516Veillonella022Anaerococcus112Peptoniphilus011Helcococcus101Staphylococcus011Turicibacter011BacteroidetesPrevotella21980198ActinobacteriaAtopobium86086Cryptobacterium27027Eggerthella21021Gardnerella707Mobiluncus505ProteobacteriaEscherichia0130Serratia2023Pseudomonas6915Janthinobacterium123Ralstonia011Dechloromonas011Klebsiella011FusobacteriaSneathia83083Total11447991943Total OTU0.033119461Typically, clones with >94% 16S rRNA gene identity to the
nearest cultivated relative were considered members of that genus.
2Clones grouped with the Prevotella genus had 92–94% 16S rRNA gene
identity to their closest cultivated relative.
3Operational
taxonomic units (OTUs) were calculated at a cutoff of 97% similarity.Among the two
participants with high-Nugent scores, the distribution ofPrevotella, Atopobium, and Cryptobacterium species was
distinct (Figure 3). A majority of Prevotella clones from participant 6 grouped with Prevotella
bivia, whereas those from participant 7 grouped most closely with Prevotella buccalis, P. corporis and P. disiens. Similarly, participant 6 had approximately three times
the number of clones that grouped with Atopobium
vaginae than did participant 7, whereas participant 7 had approximately
three times the number of clones grouped with Cryptobacterium curtum than participant 6 (Figure 3). Both
participant 6 and 7 had a similar overall distribution of species within the Megasphaera and Dialister genera.As mentioned above, clones belonging to the genusLactobacillus were the most abundant, irrespective of Nugent score.
However, participants with low-Nugent scores had a diversity of Lactobacillus species that included L. iners, L. gasseri, L. crispatus, L. jensenii, and L. vaginalis (Figure 2) whereas libraries from participants with
high-Nugent scores contained only L.
iners (Figure 2).Figure 2
Neighbor joining based phylogeny of the 1,196Lactobacillus 16S rRNA gene clones
obtained in this study. Clones that were closely related to known Lactobacillus species were condensed
into trapezia. Numbers in parentheses represent, for each group, the number of
clones obtained from participants with bacterial vaginosis (left) and without
bacterial vaginosis (right). The phylogeny is based on 503 unambiguously
aligned nucleotides. Branch points with >75% conservation are represented
with a closed circle; branch points with 50–74% conservation
are shown with an open circle. Genbank accession numbers for reference species
are shown in brackets. A 16S rRNA gene from Bacillus
subtilis was used as the outgroup. Scale bar represents 0.1 change per
nucleotide.Figure 3
Neighbor joining-based phylogeny of 16S rRNA gene clones
related to bacterial genera consistently present in participants with bacterial
vaginosis. Closely related clones were condensed into trapezia with numbers in
parentheses representing, for each group, the number of clones obtained from
participant 6 (left) and participant 7 (right). The phylogeny is based on 503
unambiguously aligned nucleotides. Branch points with >75% conservation are
represented by a closed circle. Genbank accession numbers for reference species
are shown in brackets. A 16S rRNA gene fromAquifex
pyrophilus was used as the outgroup. Scale bar represents 0.1 change per
nucleotide.


## 4. Discussion

Regarding the vaginal tract community structure of women with low-Nugent
scores, our results were similar to previously reported studies [18–20, 23, 42]. All communities were
dominated by Lactobacillus spp.  Five
different species were detected in the 758 Lactobacillus sequences including L. iners, L. gasseri,
L. crispatus, L. jensenii, and L.
vaginalis. Interestingly, we failed to detect any Lactobacillus sp. other
than L. iners in BV positive women.
Similar asymmetric distribution of lactobacillus species have been reported
where L. gasseri and L. iners were “negatively correlated to
each other” [43] or positively correlated with
BV-associated bacteria [44]. Our results suggest that L. iners may be better adapted to the
polymicrobial state of BV, including elevated pH.Bacterial vaginosis has been described as a polymicrobial syndrome [19, 21, 25, 45, 46] with higher microbial
diversity than what is perceived as the healthy ground state dominated by
lactobacilli. Clinically it is characterized by a white discharge, an increase
in pH and amine concentration, the appearance of clue cells, and a microbial
community shift detected by Gram stain of smears from vaginal fluid [19, 21, 24–26, 45, 46]. Similar to previous work (e.g.,
[19, 47]) we detected greater species
diversity in the BV positive subjects. In our 7 samples from the two BV
positive women, we detected five clades within the Prevotella genus, the most abundant of the nonlactobacillus genera
present in our libraries. Two of the Prevotella clades detected were present in both BV positive women while three were present
in only one. This may reflect host differences that select for unique species
or the consequences of sampling at nonsaturating levels. Magasphaera (2 clades, 180 clones), Dialister (2 clades, 41 clones), Cryptobacterium (1 clade, 27 clones) Atopobium (1 clade, 86 clones), Eggerthella (1 clade, 21 clones), and Gardnerella (1 clade, 7 clones) were also detected in BV positive women, although clone
numbers were different. The surprising aspect of these studies was the
volatility in clone demographics over time exhibited by the BV positive women.
This suggests that in the case of women with clinically identified BV, the
increase in diversity is accompanied by a decrease in community stability. It
is possible that in spite of the phylogenetic volatility, the community
function remains constant, as in the case of the previously cited bioreactors [17]. Other explanations are
possible (see below). Nonetheless, in our BV positive women the phylogenetic
composition changed dramatically over time in contrast to women with low-Nugent
scores.It is intriguing to consider the vaginal community in the light of
ongoing discussions of biodiversity and stability of ecosystems, in part
because of the demographic instability that we detect when the community is at
its greatest diversity, in the BV positive women. While it seems (somewhat)
intuitive to equate high biodiversity with a more resilient ecosystem; previous
workers have concluded that there was “no such arbitrarily general rule” [48, 49]. Indeed, May points out that
randomly constructed ecosystems “are more likely to lose species after
disturbances than are simple ones” [49]. Moreover, in a separate
paper May reported that simple nonlinear difference equations that describe
growth can produce stable cycles as well as apparent chaotic regimes [50]. Hence, the community
instability in the BV positive state that we observed could be more a
reflection of a randomly assembled community and/or the composite of populations
with nonoverlapping growth curves.BV can be a recalcitrant condition even in the face of clinical treatment
[25, 26, 46]. While the molecular
approaches of microbial ecology have provided considerable insight into the
phylotypes present [18–20, 23, 28, 42, 44], we remain somewhat distant
from a complete ecological description of the vaginal community that includes
the host genotypic variability, environmental influences, a complete
description of the community including eukaryotes, bacteria and viruses [51] and critical interactions
between species, not to mention prevailing nutrient sources and food webs [52]. It is encouraging that some
investigators have identified strong correlations between certain bacterial
phylotypes and BV (e.g., [19]). In addition, the hormonal
milieu appears to influence vaginal microflora, as evidenced by a lower
prevalence of BV in women exposed to exogenous hormones [31, 53] and a higher prevalence of
BV in the first week of the menstrual cycle [31, 54].
In this study of seven selected participants, we specifically chose women who
were unexposed to exogenous hormones and had consistent BV scores (i.e.,
primarily negative/intermediate or primarily positive) irrespective of the
timing in the menstrual cycle. Moreover, we have come to view the
syndrome as an ecosystem gone awry and currently efforts are being directed at
identifying the conditions or events that cause community shifts [25, 26, 46]. While this ecosystem
approach is more complex, it may prove more productive than pathogen
hunting.Our report is a preliminary study of relatively few women sampled over
time where community structure was determined using culture independent
techniques. We recognize the potential biases that can arise from PCR
amplification and library construction [55] including primer bias. The
latter is of particular concern because some phylogenetic groups can be missed
entirely by poorly matched primer sets. For example Frank et al. [56] and Verhelst et al. [42]
recently demonstrated that
detection of Gardnerella, a genus
frequently associated with BV (e.g., [42]), can be strongly influenced
by primer selection. While our primer set did pick up Gardnerella sequences, the abundance may have been influenced by
primer bias. However, in spite of these limitations we have identified
substantial diversity within the Prevotella clones, an asymmetric distribution of the lactobacilli species and large
demographic shifts over time in BV positive women.



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*Source: 181253-2009-01-27.xml*