Bacterial Communities of the Coronal Sulcus and Distal Urethra of Adolescent Males

Department of Biology, Indiana University, Bloomington, Indiana, United States of America.
PLoS ONE (Impact Factor: 3.23). 05/2012; 7(5):e36298. DOI: 10.1371/journal.pone.0036298
Source: PubMed
ABSTRACT
Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity.

Full-text

Available from: George Weinstock
Bacterial Communities of the Coronal Sulcus and Distal
Urethra of Adolescent Males
David E. Nelson
1
*, Qunfeng Dong
2.
, Barbara Van Der Pol
3.
, Evelyn Toh
1
, Baochang Fan
1
, Barry P. Katz
4
,
Deming Mi
4
, Ruichen Rong
2
, George M. Weinstock
6
, Erica Sodergren
6
, J. Dennis Fortenberry
5
*
1 Department of Biology, Indiana University, Bloomington, Indiana, United States of America, 2 Department of Biological Sciences, Department of Computer Science &
Engineering, University of North Texas, Denton, Texas, United States of America, 3 School of Public Health, Indiana University, Bloomington, Indiana, United States of
America, 4 Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 5 Department of Pediatrics, Indiana
University School of Medicine, Indianapolis, Indiana, United States of America, 6 Department of Genetics, Washington University St. Louis School of Medicine, St. Louis,
Missouri, United States of America
Abstract
Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas
altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal
taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of
these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected
from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their
urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa,
including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually
experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS
specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast,
Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine
support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be
colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity.
Citation: Nelson DE, Dong Q, Van Der Pol B, Toh E, Fan B, et al. (2012) Bacterial Communities of the Coronal Sulcus and Distal Urethra of Adolescent Males. PLoS
ONE 7(5): e36298. doi:10.1371/journal.pone.0036298
Editor: Jacques Ravel, Institute for Genome Sciences, University of Maryland School of Medicine, United States of America
Received December 14, 2011; Accepted March 30, 2012; Published May 11, 2012
Copyright: ß 2012 Nelson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health Grants NIH UH2 DK083980 to JDF and NIH RC2 HG005806 to DN and QD. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: nelsonde@indiana.edu (DEN); jfortenb@iupui.edu (JDF)
. These authors contributed equally to this work.
Introduction
The bacterial communities (microbiotas) associated with differ-
ent body surfaces can impact pathogen colonization resistance and
autoimmune disease [1]. For example, dysbioses of the gastroin-
testinal tract microbiota can trigger overgrowth of pathogens [1]
such as C. difficile [2], which are linked to chronic inflammatory
conditions including Crohn’s disease and ulcerative colitis [3,4],
and can increase risk of colonization by enteropathogens including
Clostridia, Salmonella, Vibrio, Escherichia and Shigella spp. [5,6].
Probiotic activities of Lactobacillus spp. that colonize the vagina
illustrate mechanisms by which the microbiota can influence
susceptibility to infectious disease [7]. Lactobacillus spp. regulate the
balance of pro-inflammatory cytokines in vaginal secretions
[8,9,10], block colonization and invasion of some pathogens [11]
and produce lactic acid, hydrogen peroxide [12] and bacteriocins
[13] that inhibit other vaginal microorganisms. Reduction of
vaginal Lactobacillus spp. is associated with the overgrowth of
anaerobic bacteria that occurs in bacterial vaginosis (BV) [14], and
increased susceptibility to bacterial and viral sexually transmitted
infection (STI) [15,16]. Thus there is strong evidence that the
composition of the female reproductive tract microbiota is linked
to reproductive health and resistance to STI in women.
In comparison, the microbiota of the male reproductive tract is
poorly described. The penis itself provides distinct anatomical
environments in the urethra and the coronal sulcus (CS). Both sites
are exposed to similar foreign microbial communities during
sexual activity. Some bacteria transferred during sexual activity
(e.g., Neisseria gonorrhoeae and Chlamydia trachomatis) cause substantial
world-wide morbidity [17]. In addition, the CS and distal urethras
of healthy men at least episodically support bacterial communities
[18,19,20,21]. Lactobacillus spp. have been identified in urine and
urethral swabs [19], and BV-associated taxa including Prevotella,
Gardnerella and Sneathia are found in CS [20] and urethral
specimens from adult men [18,19]. Although the role of bacteria
in the male urethra is unknown, the CS microbiota has been
hypothesized to mediate effects of circumcision on risk of HIV and
other STI [20].
A limitation in understanding the microbiota of the penis is the
lack of data from healthy young men who have and or have not
had partnered sexual experiences. These data would allow more
thorough description of the microbiota of the urethra and CS, and
could provide insight into changes associated with sexual
PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36298
Page 1
exposures. To fill this gap, we collected urine and CS specimens
from eighteen healthy 14–17 year old men with varied circum-
cision status and sexual histories. Sampling was repeated at
monthly intervals to investigate stability of the microbiota over a
three-month period. Bacteria were identified using multiple 16 S
rRNA sequencing methods. Urine and corresponding CS speci-
mens supported stable, but dissimilar microbiotas. Major urine
taxa in most of the sexually experienced and inexperienced
participants were members of the order Lactobacillialles. Finally,
some bacteria were detected only in participants with histories of
partnered sexual activity.
Results and Discussion
Compositions of CS and urine microbiotas are similarly
measured by different 16 S rRNA sequencing approaches
There were no data concerning optimal methods for cultivation-
independent characterization of CS or urine microbiotas; thus we
analyzed total genomic DNA from 72 CS and urine specimens
using four different 16 S rRNA sequencing approaches. Near full-
length 16 S rRNA alleles were assembled from individual Sanger
sequence reads [18], while the V1–V3, V3–V5 and V6–V9 sub-
regions of 16 S rRNA alleles were analyzed by multiplex PCR and
pyrosequencing using a protocol developed by human microbiome
demonstration projects (http://hmpdacc.org) [22]. Only 8 urine
and 3 CS specimens yielded poor quality or no PCR amplicons
using the Sanger approach and these same specimens tended to
yield poor pyrosequencing results. This indicated that some
specimens either had low bacterial loads or contained inhibitors
that blocked 16 S PCR amplification. Taxonomy was assigned
using RDP classifier. In total, 56,406; 756,640; 759,914 and
1,212,918 sequences from full-length, V1–V3, V3–V5 and V6–V9
16 S rRNA regions were assigned to the genus level with 90%
confidence, respectively (Tables S1, S2, S3, S4, S5).
PCR-based 16 S rRNA identification can introduce biases
during priming, amplification, cloning and sequencing [23]. Types
and proportions of taxa identified in urine and CS specimens by
each sequencing approach were compared. Major taxa and their
relative proportions were similar in all the datasets (Figures 1a and
1b; Tables S1, S2, S3, S4, S5) with the following exceptions: V6–
V9 under-represented Prevotella, Gardnerella was not captured by the
V1–V3 approach, and Corynebacteria were over-represented in both
V1–V3 and V3–V5. These results indicated that sub-region
sequencing provided reasonable coverage of both urethral and CS
bacterial communities, with the caveat that validation against
other methods is warranted to reveal taxa missed by any single
method. Since a large number of near full-length Sanger 16 S
sequences were obtained and pyrosequencing failed to reveal
additional major taxa our subsequent analyses were based on the
Sanger data and cross-checked against the sub-region data sets.
The CS Supports a Complex and Stable Microbiota
In the 17/18 enrollment specimens that yielded 16 S rRNA
amplicons, 58 high confidence taxa were predicted from 9,070
16 S rRNA sequences (Figure 2A). Three genera were in most
specimens; Corynebacteria (16/17), Staphylococcus (16/17) and Anaero-
coccus (15/17), and these accounted for more than 58.9% of the
sequences. Other abundant genera, in order of relative abun-
dance, included Peptoniphilus (13/17), Prevotella (4/17), Finegoldia
(14/17), Porphyromonas (8/17), Propionibacterium (11/17) and Delftia
(8/17). All of these genera, along with high quality sequences that
could not be classified to the genus-level with 90% confidence
(11.2%), accounted for an additional 30.5% of the sequences
(Figure 2A and Table S1). Pseudomonas were notably less abundant
(,0.01%) in CS specimens in this cohort than they were in a
group of adult African men described in a previous study [20].
Comparison of major taxa in enrollment and subsequent
monthly CS specimens indicated no dramatic differences in
composition during the study interval. To assess stability of the CS
microbiota, weighted and unweighted Unifrac PCA [24], the
Sørenson similarity index and Spearman correlation coefficient
were used to compare longitudinal CS specimens. All of these
measures confirmed that longitudinal CS specimens from the same
participant were significantly more similar to one another than to
CS specimens from other participants (Figure 3A). Lin’s concor-
dance correlation coefficents were calculated to measure stability
of individual taxa (Figure 4A). Staphyloccoccus, Mobiluncus, Prevotella,
Dialister and Anaerococcus yielded mean values of . 0.5, indicating
that these taxa were stable members of CS communites. In
contrast, major urine taxa including Veillonella and Streptococcus
were detected in some CS specimens but yielded values between 0
and 0.25. This indicates that although some urine taxa periodically
colonize or reside in the CS, they are not stable members of the
CS microbiota.
Circumcision alters the CS microbiota
Price and colleagues reported that circumcision alters the adult
CS microbiota and that anaerobic and putative vaginal taxa are
most abundant prior to circumcision, whereas aerobes and skin
taxa increase following circumcision [20]. Taxa in enrollment
specimens from participants who had (12/17) and who had not (5/
17) been circumcised were compared to test if similar relationships
could be detected in young men (Figure 5A). Consistent with the
Price study, Corynebacteria and Anaerococcus were dominant in both
groups and were present in similar proportions. Staphylococcus was
significantly enriched (26.6% vs. 5.5%) in enrollment specimens
from circumcised participants (Wilcoxon signed-rank test, p-
value = 0.0048) while Porphyrmonas was enriched (6.4% vs. 0.3%)
in uncircumcised participants (Wilcoxon signed-rank test, p-
value = 0.026). Prevotella was exclusively identified in uncircumcised
participants (4/6) (Fisher’s exact test p-value = 0.006) and was
abundant in this sub-group (12.9%).
Weighted Unifrac analysis robustly separated CS and urine
specimens (Figure 6A). This analysis also clearly differentiated
circumcised and uncircumcised CS specimens (Figure 6B). In
contrast, groups of circumcised and uncircumcised urines were not
as clearly differentiated (Figure 6C). Clustering of CS and urine
CS specimens from each sampling point based upon Bray-Curtis
and Sørenson’s similarity coefficients as a measure of distance also
indicated that CS and urine specimens from the same subject
rarely grouped together (Figure S1 and data not shown).
Collectively, these results indicated that circumcision most strongly
impacted the CS and that the microbiotas in corresponding CS
and urine specimens were dissimilar.
Circumcised and uncircumcised CS contained taxa that have
been associated with the superficial skin [25] and especially moist
skin sites, such as the inguinal crease [26]. CS contained high
proportions of Corynebacteria or Staphyloccocus and lower proportions
of Propionibacteria and Betaproteobacteria; patterns associated with
sebaceous and dry skin sites, respectively [26]. Some taxa
including Prevotella and Porphyromonas were more abundant in
uncircumcised CS, but were not predominant components of the
CS microbiota. Interestingly, multiple taxa common in urine were
rare or absent from CS specimens.
Microbiotas of Urine and CS are Dissimilar
18/18 enrollment urines yielded specific PCR amplicons and 51
high confidence taxa were predicted from a total of 10,144 16 S
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36298
Page 2
Figure 1. Comparison of CS and urine microbiotas measured by different 16 S rRNA sequencing methods. Distribution of RDP taxa
(90% confidence) in enrollment (A) CS and (B) urine specimens. Proportions of normalized sequences are on the Y-axis. (black) Sanger, (white) V1–V3,
(dark gray) V3–V5 and (light gray) V6–V9 sequence data sets.
doi:10.1371/journal.pone.0036298.g001
Figure 2. Distribution of major taxa in enrollment CS and urine specimens. Sanger data-set. (A) CS specimens. (B) urine specimens.
doi:10.1371/journal.pone.0036298.g002
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36298
Page 3
rRNA sequences. Some abundant CS taxa were detected in urine
but microbiotas of these specimen types were dissimilar (Figure 2).
Urine contained high proportions of genera whose members are
obligate and/or facultative anaerobes (Figure 2B). Streptococcus (11/
18), Lactobacillus (4/18), Gardnerella (5/18) and Veillonella (4/18),
accounted for 59.1% of enrollment urine sequences. Closest RDP
matches of almost all of the enrollment Lactobacillus and Gardnerella
sequences corresponded to L. iners and G. vaginalis, while more
diverse species of Streptococcus (agalactiae, mitis, anginosus and
uncultured taxa) and Veillonella (dispar, atypica, montpellierensis, parvula
and uncultured taxa) were represented. In contrast, only 1.3% of
CS sequences corresponded to these taxa and .99% of these were
Streptococcus.
The urine Microbiota is Less stable than is the Microbiota
of the CS
Similar to CS, independent measures confirmed urine speci-
mens from the same participants were significantly more similar to
one another than urine specimens from other participants
(Figure 3B). To compare relative stability of the CS and urine
microbiotas, Sørensen’s similarity coefficients were computed for
pairwise specimens from different sampling points for each subject
swab and urine specimen. The analysis incorporated a linear
mixed-effect model to account for clustering due to multiple
specimens from each subject (Figure 7). Sørensen’s similarity
coefficient of CS (0.60) was significantly higher than urine (0.52)
(p-value = 0.0001) indicating that the CS microbiota was more
stable.
Some urine taxa were stable during the study period (Figures 4B
and 5B). Average lengths of continuous colonization with
Propionibacterium and Lactobacillus were 2.33 and 2.75 months
respectively, and Lin’s concordance correlation coefficent for these
taxa were .0.5. Outside these ‘‘core’’ taxa, the urine microbiota
was less stable than CS. Corynebacteria, Anaerococcus, Staphylococcus
and Prevotella accounted for only 22.1% of enrollment urine
sequences as opposed to 63.5% of CS sequences (Figure 2).
Average lengths of continuous colonization with these taxa were
all ,1.5 months and Lin’s concordance correlation coefficents
were all ,0.5 (Figure 4B). When present in sequential urine from
Figure 3. Comparison of intraperson and interperson similarity of CS and urine microbiotas. Black bars indicate comparison of
specimens from the same participant (4 specimens) and white bars indicate comparison of each specimen to all specimens from different
participants. A) CS specimens. B) Urine specimens. SO, Sørenson similarity index; SC, Spearman similarity coefficient; UU, unweighted Unifrac distance;
WU, weighted Unifrac distance. Wilcoxon P-values for all comparisons (same versus different participants) were all ,1E10
26
. Bars indicate 95%
confidence intervals.
doi:10.1371/journal.pone.0036298.g003
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36298
Page 4
the same participant the relative abundance of these taxa also
varied widely (data not shown). Our interpretation is that some of
these taxa may not be true urethral residents but contaminants
from the urethral meatus or CS.
The Mic robiota of Urine is Simila r to that of other
Mucosal Surfaces
Additional results supported the hypothesis that microbial
communities of the urethra and CS are distinct. Lactobacillus,
Veillonella, Aerococcus, Ureaplasma, Gardnerella and Mycoplasma were
detected only, or almost exclusively, in urine. Bacteria in these
genera have been identified in mucosal samples of which
Ureaplasma, Gardnerella and Mycoplasma are urogenital pathogens
[27,28].
Partnered sexual exposures impact the penis microbiota
Comparison of the microbiotas of young men in this study with
older men from previous studies [18,19,20] suggested sexual
history could be a determinant of the penis microbiota compo-
sition. Known sexually transmitted organisms (N. gonorrhoeae, C.
trachomatis, U. parvum and M. genitalium) and other poorly
characterized taxa associated with the urogenital tract in other
studies (M. hominis and Sneathia spp.) were rare or not detected in
this study. Because different types of sexual activities expose the
CS and urethra to different microbiotas we reasoned that they
might also impact penis microbiotas differently.
Only one participant reported any history of insertive anal sex,
whereas 11/18 (61%) reported ever having had vaginal sex and
10/18 (56%) reported fellatio. Lactobacillus, Prevotella, Streptococcus,
Staphylococcus and Anaerococcus were detected at similar frequencies
in urine and CS from participants with and without sexual
experience. BV-associated taxa including Atopobium, Megasphaera,
Mobiluncus, Prevotella, Gemella, Veillonella, Gardnerella and Clostridium
[29] were each detected in one or more participants who reported
no partnered sexual experiences (Tables S1, S2, S3, S4). These
results indicate many putative vaginal taxa also colonize the male
urethra and CS, possibly from other body sites or environmental
reservoirs. Alternatively, urine from male infants frequently
contain Lactobacillus [30] so these could be vertically inherited
Figure 4. Temporal stability of CS and urine taxa. Lin’s concordance correlation coefficients (Y-axis) were calculated to assess agreement of
abundance of taxa, (X-axis), in sequential (A) CS, or (B) urine specimens from the same participants (three intervals: months 0–1, 1–2, 2–3), the red
trend line indicates mean of the three intervals. Bars indicate 95% confidence intervals.
doi:10.1371/journal.pone.0036298.g004
Figure 5. Impact of circumcision on the CS and urine microbiotas. Relative normalized abundance of major (A) CS and (B) urine taxa in
circumcised (red) and uncircumcised (blue) participants at all four sampling points (Z-axis). Bars indicate 95% confidence intervals.
doi:10.1371/journal.pone.0036298.g005
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36298
Page 5
communities. In either scenario, the observation that the male and
female genital tracts support similar microbiotas suggests that
some of these organisms could survive sexual transfer.
Colonization rates in asymptomatic older men at high risk for
STI with various Ureaplasma, Mycoplasma and Sneathia spp. can
exceed 30% [18,19]. In this study, Ureaplasma, Mycoplasma, and
Figure 6. Similarity of CS and urine taxa. A-C) Weighted Unifrac comparison of the microbiotas in select groups of specimens (Sanger data-set).
A) All CS (red) and all urine (blue) specimens. B) Circumcised (red) and uncircumcised (blue) CS specimens. C) Circumcised (red) and uncircumcised
(blue) urine specimens.
doi:10.1371/journal.pone.0036298.g006
Figure 7. The microbiota of the CS is more stable than that of urine. Sørenson’s similarity indices calculated between pairwise specimens
within each participant’s (month = 0, 1, 2, 3) CS and urine samples, separately. A linear mixed-effects model was used to test if the index differed
between CS and urine samples, taking into account multiple specimens from the same participants (clustered data). Participants are indicated at left,
Sørenson similarity values are on the Y-axis, and blue dots indicate unique specimens.
doi:10.1371/journal.pone.0036298.g007
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36298
Page 6
Sneathia were each detected in two participants (considering all sets
of sequence data and excluding singlets in 454 data-sets that could
result from rare miscalls of primer bar codes [31]) and one
participant was co-colonized with all three organisms. All of these
participants reported sexual exposures. Interestingly, Sneathia was
detected in one participant co-incident with his first anal sexual
exposure. These observations support the hypothesis that sexual
exposures could alter the composition of the male urethral flora.
Similarities of urine and vaginal microbiota
Only 2 enrollment urines lacked Lactobacillus and/or Streptococcus
entirely. One contained a high proportion of unclassified (20.0%)
and Escherichia coli (36.9%) sequences, indicating possible urinary
tract infection. The other contained a mixture of Prevotella and
Staphylococcus. All other enrollment urine contained high proportions
of Streptococcus or Lactobacillus, but rarely both. Spearman’s rank
correlation coefficient was used to assess the co-occurrence of taxa in
urine specimens. The occurrences of Lactobacillus and Streptococcus in
urine specimens were negatively correlated (r = 20.40, p-val-
ue = 0.0012). Interestingly, similar Lactobacillus- and Streptococcus-
dominated communities can maintain the low pH of the vagina [32].
Our results show that the penis does not support a single
characteristic microbiota and that it is important to distinguish
between the urethra and CS in understanding the compositions
and possible interactions of these microbiotas with STI. Sexually
transmitted pathogens that colonize the penis can exclusively
colonize the urethra (C. trachomatis, N. gonorhoeae, M. genitalium and
U. urealyticum) or both the urethra and CS (human papilloma virus,
herpes simplex virus (HSV-2), human immunodeficiency virus
(HIV), H. ducreyi, T. pallidum). Whether the CS and urethral
microbiota contribute to STI colonization resistance is unclear but
reports that circumcision differentially impacts risk for HIV [33],
HSV-2, T. pallidum and H. ducreyi [34] and urethral pathogens (C.
trachomatis and N. gonorhoeae) [35] is in concordance with our
findings. A future direction would be to test if the urethral
microbiota is relevant to risk for urethral STI.
It has long been appreciated that taxa that resemble those in the
female genital tract, colonize the male urethra, at least episodically
[21]. Our results are the first to show that some of these taxa
persist and colonize the penis prior to the onset of partnered sexual
activity. Colonization of the female and male genital tracts with
similar bacteria may reflect shared embryonic origins of these
tissues. It also suggests that these microbiotas might have common
ecological functions, such as lactic acid production and pathogen
resistance. A caveat is that 16 S rRNA sequencing does not yield
sufficient resolution to differentiate if the taxa in male and female
genital tract specimens are indeed the same organisms or tell us if
they have similar metabolic capacities.
Finally, our results suggest that partnered sexual behavior can
alter the composition of the urogenital microbiota. Although small
sample size limits the confidence of some conclusions, it is still
noteworthy that BV associated taxa including Mycoplasma,
Ureaplasma, and Sneathia were detected only in sexually experienced
participants [29]. Technologies that differentiate strain-level
polymorphisms, such as metagenomic sequencing, could be used
to test if these organisms are sexually transmitted and could yield
insights into the nature of idiopathic urogenital tract syndromes
that impact both men and women.
Methods
Study Design
Young men (ages 14–17 at enrollment; N = 18) were enrolled as
a single cohort during the week of January 2, 2010. The
participants were a non-clinical sample, recruited from diverse
community contacts, although not all are resident in a single
community. Additional enrollment criteria included no antibiotic
use in the past 60 days, absence of existing urinary tract infection
(including sexually transmitted infections), absence of immune
suppressing conditions, and overall good general health status.
Prior sexual activity was not considered. Self-reported race/
ethnicity was White (7/18), Black (7/18), and Latino (4/18).
Self-reported behavioral data was collected at enrollment using
a web-based, computer-assisted self-interview. Participants report-
ed circumcision status, lifetime and recent sexual activity (oral,
anal, vaginal), and presence or absence of genitourinary symp-
toms. All participants were asymptomatic at enrollment. All
participants and parents provided written, informed consent.
Ethics approval for this study was obtained from the Institu-
tional Review Board of Indiana University and all subjects were
recruited through the Indiana University Medical School. Written
informed consent to participate in the study was provided by next
of kin, caregivers or guardians on the behalf of the minors/
children participants in this study and this procedure was
approved by the Institutional Review Board of Indiana University.
Specimens were collected at enrollment, and at three subse-
quent one-month intervals. Specimens were collected in the
participant’s home. Voided urine sample and a CS swab sample
were collected at each time point. Urine was obtained by first-
catch void into a sterile collection cup. CS samples were obtained
following training on a flaccid penis model. Participants were
instructed to retract the foreskin (if present), and firmly trace the
groove of the coronal sulcus circumferentially (using a flocked
4 mm flexible handle elution swab). Samples were immediately
placed in a cooler, and transported to the laboratory within four
hours were they were stored at 280 C until DNA extraction.
Molecular Methods
CS samples were thawed and then suspended in 1 ml PBS then
were vortexed for 1 min. Urine (10 ml) were thawed and pelleted
by centrifugation for 15 min at 4,0006g at 4uC. In both cases,
DNA was harvested from specimens using a Qiagen DNeasy
(Qiagen Inc., Valencia CA) blood and tissue extraction kit.
Genomic DNA was eluted in nuclease-free water and stored at
4uC until sequencing. Reagent only controls were processed in
parallel to monitor for reagent contamination [18]. 16 S rRNA
PCR, Sanger sequencing and assembly of sequences were
performed as described previously [19]. Pyrosequencing of the
V1–V3, V3–V5 and V6–V9 sub-regions was performed according
a protocol developed by the Human Microbiome Project Data
Generation Working Group available at http://www.hmpdacc.
org [22].
Sequence Analysis and Statistics
16 S sequences were processed as described previously (18, 19).
Briefly, raw 16 S sequence reads were compared to human
genome sequences at NCBI using BLASTN [36]. Sequence reads
below the BLAST E-value cutoff 10
–20
were considered putative
human DNA contaminants and were removed from the subse-
quent analyses. For 16 S rRNA contigs obtained from Sanger
sequence reads, we applied the GreenGenes criteria to define full-
length or near-full 16 S gene (i.e., the sequence length
. = 1250 bp). Only full length or near-full length contigs were
selected for subsequent analysis. These contigs were screened for
chimeras using Bellerophon [37] and default parameters at the
GreenGenes web server [38]. For sequence reads obtained from
454 sequencing technology, each read was sorted to specimens
only if a perfect barcode sequence was detected. Sequences that
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36298
Page 7
did not contain perfect matches to primer barcodes, were less than
200 bp in length, and or had average quality scores of less than 25
were discarded. The primer and barcode sequences were then
trimmed from the remaining sequences using customized Perl
scripts. Both the 16 S contigs from Sanger sequencing and 16 S
raw sequences were classified with RDP Classifier v2.2 [39] using
a series of confidence cutoffs ranging from 0.6 to 0.9. For selected
subset of genera member sequences were BLASTed against the
SILVA database [40]. Species-level assignments were decided
based on near perfect alignments to top database matches and
were manually examined for alignment quality (i.e., both
alignment length and percentage similarity). Multiple sequence
alignments were produced using ClustalW [41] with default
parameters, and neighbor-joining phylogenic trees were construct-
ed using PHYLIP [42]. Principal coordinate analyses of microbial
communities were performed using Unifrac [24]. The above
Unifrac analyses were also repeated by random sub-sampling from
each specimen. Briefly, sequence reads were randomly extracted
without replacement from each specimen (100 reads for Sanger
sequences and 200 reads for 454 sequences) and this procedure
was repeated 100 times. The averaged sequence counts were used
for Unifrac analyses. Microsoft EXCEL and customized scripts
developed in the R statistical package (http://www.r-project.org/)
were used for statistical analyses. To correct for multiple tests, false
discovery rates were computed with the R package function
qvalue. Wilcoxon’s rank sum test was used to assess comparisons of
continuous variables between groups. The Wilcoxon’s signed rank
test for paired data was used for comparisons between swab and
urine samples from the same subject. Fisher’s exact test was used
for comparisons of categorical variables between groups. Sør-
ensen’s similarity index was calculated between either swab or
urine specimens from different time points within each subject to
assess intra-subject variability of microbiotas over time. Differenc-
es in the index between swab and urine samples were assessed
using linear mixed-effects models. Lin’s concordance correlation
coefficent was used to assess the agreement of measurements of
abundance of different taxa over time. A 95% confidence interval
was also calculated for Lin’s concordance correlation coefficient.
Spearman’s correlation coefficent was employed to assess whether
the abundances of two taxa were independent or associated. P
values less than 0.05 were considered significant. All sequences
from this study are available at www.microbiota.org/mum.htm.
Supporting Information
Figure S1 Heirarchical clustering of enrollment urine
and CS swab microbiomes. Sanger sequences from enroll-
ment urine and swab specimens were heirarchically clustered
using A) Bray-Curtis and B) Spearman’s correlation coefficients as
a measure of distance. Urine and swab specimens are labeled U
and S, respectively.
(TIF)
Table S1 RDP classifier summary of 16 S rRNA se-
quences and select meta-data. Tables list 90% confidence
RDPII results for quality-checked sequences. Sanger data set.
(XLS)
Table S2 RDP classifier summary of 16 S rRNA se-
quences and select meta-data. Tables list 90% confidence
RDPII results for quality-checked sequences. V1–V3 data-set.
(XLS)
Table S3 RDP classifier summary of 16 S rRNA se-
quences and select meta-data. Tables list 90% confidence
RDPII results for quality-checked sequences. V3–V5 data-set.
(XLS)
Table S4 RDP classifier summary of 16 S rRNA se-
quences and select meta-data. Tables list 90% confidence
RDPII results for quality-checked sequences. V6–V9 data-set.
(XLS)
Table S5 Numbers and sizes of 16 S rRNA amplicons by
sample type and method.
(DOC)
Author Contributions
Conceived and designed the experiments: DEN BVDP QD GMW ES
BPK JDF. Performed the experiments: JDF BVDP BF ET GMW ES.
Analyzed the data: DEN BVDP BPK DM JDF RR QD. Wrote the paper:
DEN JDF.
References
1. Frank DN, Zhu W, Sartor RB, Li E (2011) Investigating the biological and
clinical significance of human dysbioses. Trends Microbiol.
2. Chang JY, Antonopoulos DA, Kalra A, Tonelli A, Khalife WT, et al. (2008)
Decreased diversity of the fecal Microbiome in recurrent Clostridium difficile-
associated diarrhea. J Infect Dis 197: 435–438.
3. Frank DN, St Amand AL, Feldman RA, Boedeker EC, Harpaz N, et al. (2007)
Molecular-phylogenetic characterization of microbia l community imbalances in
human inflammatory bowel diseases. Proc Natl Acad Sci U S A 104:
13780–13785.
4. Eckburg PB, Relman DA (2007) The role of microbes in Crohn’s disease. Clin
Infect Dis 44: 256–262.
5. Stecher B, Chaffron S, Kappeli R, Hapfelmeier S, Freedrich S, et al. (2010) Like
will to like: abundances of closely related species can predict susceptibility to
intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog 6:
e1000711.
6. Stecher B, Hardt WD (2008) The role of microbiota in infectious disease. Trends
Microbiol 16: 107–114.
7. Servin AL (2004) Antagonistic activities of lactobacilli and bifidobacteria against
microbial pathogens. FEMS Microbiol Rev 28: 405–440.
8. Witkin SS, Linhares IM, Giraldo P, Ledger WJ (2007) An altered immunity
hypothesis for the development of symptomatic bacterial vaginosis. Clin Infect
Dis 44: 554–557.
9. Donders GG, Vereecken A, Bosmans E, Spitz B (2003) Vaginal cytokines in
normal pregnancy. Am J Obstet Gynecol 189: 1433–1438.
10. Mirmonsef P, Gilbert D, Zariffard MR, Hamaker BR, Kaur A, et al. (2011) The
effects of commensal bacteria on innate immune responses in the female genital
tract. Am J Reprod Immunol 65: 190–195.
11. Spurbeck RR, Arvidson CG (200 8) Inhibition of Neiss eria gonorrhoeae
epithelial cell interactions by vaginal Lactobacillus species. Infect Immun 76:
3124–3130.
12. Atassi F, Servin AL (2010) Individual and co-operative roles of lactic acid and
hydrogen peroxide in the killing activity of enteric strain Lactobacillus johnsonii
NCC933 and vaginal strain Lactobacillus gasseri KS120.1 against enteric,
uropathogenic and vaginosis-associated pathogens. FEMS Microbiol Lett 304:
29–38.
13. Boris S, Barbes C (2000) Role played by lactobacilli in controlling the population
of vaginal pathogens. Microbes Infect 2: 543–546.
14. Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, et al.
(1989) Prevalence of hydrogen peroxide-producing Lactobacillus species in
normal women and women with bacterial vaginosis. J Clin Microbiol 27:
251–256.
15. Schwebke JR (2005) Abnormal vaginal flora as a biological risk factor for
acquisition of HIV infection and sexually transmitted diseases. J Infect Dis 192:
1315–1317.
16. Schwebke JR, Desmond R (2007) A randomized trial of metronidazole in
asymptomatic bacterial vaginosis to p revent the acquisition of sexually
transmitted diseases. Am J Obstet Gynecol 196: 517 e511–516.
17. Starnbach MN, Roan NR (2008) Conquering sexually transmitted diseases. Nat
Rev Immunol 8: 313–317.
18. Nelson DE, Van Der Pol B, Dong Q, Revanna KV, Fan B, et al. (2010)
Characteristic male urine microbiomes associate with asymptomatic sexually
transmitted infection. PLoS One 5: e14116.
19. Dong Q, Nelson DE, Toh E, Diao L, Gao X, et al. (2011) The microbial
communities in male first catch urine are highly similar to those in paired
urethral swab specimens. PLoS One 6: e19709.
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36298
Page 8
20. Price LB, Liu CM, Johnson KE, Aziz M, Lau MK, et al. (2010) The effects of
circumcision on the penis microbiome. PLoS One 5: e8422.
21. Bowie WR, Pollock HM, Forsyth PS, Floyd JF, Alexander ER, et al. (1977)
Bacteriology of the urethra in normal men and men with nongonococcal
urethritis. J Clin Microbiol 6: 482–488.
22. Group. JCHMPDGW (2012) Evaluation of 16 S rDNA-based community
profiling for human microbiome research.
23. Rajendhran J, Gunasekaran P (2010) Microbial phylogeny and diversity: Small
subunit ribosomal RNA sequence analysis and beyond. Microbiol Res.
24. Lozupone CA, Hamady M, Kelley ST, Knight R (2007) Quantitative and
qualitative beta diversity measures lead to different insights into factors that
structure microbial communities. Appl Environ Microbiol 73: 1576–1585.
25. Gao Z, Tseng CH, Pei Z, Blaser MJ (2007) Molecular analysis of human
forearm superficial skin bacterial biota. Proc Natl Acad Sci U S A 104:
2927–2932.
26. Grice EA, Kong HH, Conlan S, De ming CB, Davis J, et al. (2009)
Topographical and temporal diversity of the human skin microbiome. Science
324: 1190–1192.
27. Taylor-Robinson D, Jensen JS (2011) Mycoplasma genitalium: from Chrysalis to
Multicolored Butterfly. Clin Microbiol Rev 24: 498–514.
28. Harwich MD, Jr., Alves JM, Buck GA, Strauss JF, 3rd, Patterson JL, et al.
(2010) Drawing the line between commensal and pathogenic Gardnerella
vaginalis through genome analysis and virulence studies. BMC Genomics 11:
375.
29. Fredricks DN, Fiedler TL, Marrazzo JM (2005) Molecular identification of
bacteria associated with bacterial vaginosis. N Engl J Med 353: 1899–1911.
30. Lee JW, Shim YH, Lee SJ (2009) Lactobacillus colonization status in infants with
urinary tract infection. Pediatr Nephrol 24: 135–139.
31. Lennon NJ, Lintner RE, Anderson S, Alvarez P, Barry A, et al. (2010) A
scalable, fully automated process for construction of sequence-ready barcoded
libraries for 454. Genome Biol 11: R15.
32. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, et al. (2010) Microbes and
Health Sackler Colloquium: Vaginal microbiome of reproductive-age women.
Proc Natl Acad Sci U S A.
33. Cameron DW, Simonsen JN, D’Costa LJ, Ronald AR, Maitha GM, et al. (1989)
Female to male transmission of human immunodeficiency virus type 1: risk
factors for seroconversion in men. Lancet 2: 403–407 .
34. Weiss HA, Thomas SL, Munabi SK, Hayes RJ (2006) Male circumcision and
risk of syphilis, chancroid, and genital herpes: a systematic review and meta-
analysis. Sex Transm Infect 82: 101–109; discussion 110.
35. Mehta SD, Moses S, Agot K, Parker C, Ndinya-Achola JO, et al. (2009) Adult
male circumcision does not reduce the risk of incident Neisseria gonorrhoeae,
Chlamydia trachomatis, or Trichomonas vaginalis infection: results from a
randomized, controlled trial in Kenya. J Infect Dis 200: 370–378.
36. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25: 3389–3402.
37. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect
chimeric sequences in multiple sequence alignments. Bioinformatics 20:
2317–2319.
38. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, et al. (2006)
Greengenes, a chimera-checked 16 S rRNA gene database and workbench
compatible with ARB. Appl Environ Microbiol 72: 5069–5072.
39. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, et al. (2007)
The ribosomal database project (RDP-II): introducing myRDP space and
quality controlled public data. Nucleic Acids Res 35: D169–172.
40. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, et al. (2007) SILVA: a
comprehensive online resource for quality checked and aligned ribosomal RNA
sequence data compatible with ARB. Nucleic Acids Res 35: 7188–7196.
41. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, et al. (2003) Multiple
sequence alignment with the Clustal series of programs. Nucleic Acids Res 31:
3497–3500.
42. Felsenstein J (1989) PHYLIP- Phylogeny ingerence package. Cladistics. pp
164–166.
Penis Microbiomes of Adolescent Males
PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e36298
Page 9
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: The RDP Classifier is a widely used bioinformatic program that performs taxonomic classification of 16S rRNA gene sequences. However, the accuracy of the program is not clear when it is applied to common PCR products of the 16S rRNA variable regions, which are heavily used in microbiome projects. In this study, full-length 16S rRNA gene alignments from the SILVA database were used to simulate the PCR products of the combined variable regions (i.e., V1V3, V3V5, and V6V9). The classification accuracies obtained from RDP Classifier were evaluated for each of the simulated 16S rRNA regions, at different confidence score thresholds. Although minor bias was observed, the RDP Classifier achieved overall similar and accurate classification results when using the combined variable regions of the 16S rRNA gene, i.e., V1V3, V3V5, and V6–V9. Additional analysis showed that V2 and V4 were the most accurate among individual regions (i.e., V1 to V9). http://www.ashdin.com/journals/MG/235551.pdf
    Full-text · Article · Mar 2012 · Metagenomics
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: This manuscript describes the male genital tract microbiota and the significance of it on the host's and his partner's health. Microbiota exists in male lower genital tract, mostly in urethra and coronal sulcus while high inter-subject variability exists. Differences appear between sexually transmitted disease positive and negative men as well as circumcised and uncircumcised men. Upper genital tract is generally germ-free, except in case of infections. Prostatitis patients have frequently abundant polymicrobial communities in their semen, expressed prostatic secretion and/or post-massage urine. Coryneform bacteria have ambivalent role in male urogenital tract being frequently commensals but sometimes associated with prostatitis and urethritis. Interactions between male and female genital tract microbiota are highly likely yet there are very scarce studies on the couples' genital tract microbiota. Increase of bacterial vaginosis-type microbiota and coliforms are the most typical findings in men while the adverse effect of male genital tract bacteria on in vitro fertilization and pregnancy outcome has also been indicated.
    Full-text · Article · Nov 2012 · Pharmacological Research
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Background. Bacterial vaginosis (BV) recurrence following recommended therapies is common, yet whether recurrence is due to persistent infection or re-infection is unknown. Our aim was to determine behaviours associated with BV recurrence in women enrolled in a randomized-controlled trial.Methods. Symptomatic 18-50 year old females with BV (≥3Amsel's criteria and Nugent score (NS)=4-10) were enrolled in a 3-arm randomised double-blind placebo-controlled trial at Melbourne Sexual Health Centre, Australia, 2009-10. 450 participants received 7-days of oral metronidazole, were equally randomised to: vaginal-clindamycin, a lactobacillus vaginal-probiotic or vaginal-placebo, and completed a questionnaire. At 1,2,3 & 6-months, participants self-collected vaginal smears and completed questionnaires. Primary endpoint was NS=7-10. Cox regression was used to estimate hazard ratios (HR) for risk of BV recurrence associated with baseline and longitudinal characteristics, allowing for repeated measures from participants and stratifying for treatment.Results. 404 (90%) women who provided post-randomization data were included in analyses. Cumulative 6-month BV recurrence was 28% (95%CI 24-33%) and not associated with treatment. After adjustment for frequency of sex and age, BV recurrence was associated with having the same pre/post-treatment regular sexual partner(RSP) (Adjusted HR=1.9;95%CI 1.2-3.0), inconsistent condom use (AHR=1.9;1.0-3.3), and being born outside Australia (AHR=1.5;1.0-2.1); use of an oestrogen-containing contraceptive was protective (AHR=0.5;0.3-0.8).Conclusions. BV recurrence was significantly increased by remaining with the same pre/post-treatment RSP and inconsistent condom use, and halved with use of oestrogen-containing contraceptives. Behavioural and contraceptive practices appear to play a significant role in modifying the effectiveness of antibiotic therapies in the treatment of BV. These findings have implications for clinical practice.
    Preview · Article · Dec 2012 · Clinical Infectious Diseases
Show more