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The New World of the Urinary Microbiome in Women



Emerging evidence challenges the long-held paradigm that the healthy bladder is sterile. These discoveries may provide new opportunities to address important women’s health conditions, including pre-term labor and delivery, urinary tract infections and common forms of urinary incontinence. Traditional tools for urinary bacterial assessment, including urinary dipsticks and standard urine cultures, have significant limitations that restrict the information available to clinicians. For example, the standard urine culture does not detect slow growing bacteria that die in the presence of oxygen. Two new, complementary tools, however, can detect these and other organisms, permitting a more complete characterization of bacterial communities within the female bladder. Obstetrician-gynecologists should become familiar with these new approaches (expanded quantitative urine culture and 16S rRNA gene sequencing), which can detect previously unrecognized organisms. These advances are making it possible to answer previously intractable scientific and clinical questions.
The new world of the urinary microbiota in women
Linda Brubaker, MD, MS; Alan J. Wolfe, PhD
For decades, clinicians have used a
small set of tools to assess the bac-
terial milieu within the bladder. These
tools have included ofce-based urinary
dipsticks, formal urinalyses, and stan-
dard urine cultures to rule out the
presence of uropathogens that are
responsible for conditions such as uri-
nary tract infection (UTI) and the
less well-understood phenomenon of
asymptomatic bacteriuria. New discov-
eries made with novel methods have
highlighted the limitations of this tradi-
tional toolkit, unveiled problems with
our nomenclature, and revealed aws
in our assumptions. In this Clinical
Opinion, we will describe the limitations
of current testing and the difculties
with current nomenclature. We will
present some new techniques and
their associated scientic nomenclature.
Because these new approaches may
soon enter the clinical care algorithm,
we will describe how the new data are
derived and displayed. Finally, we will
present some of the new ndings. Our
goal is to equip practicing clinicians
with the concepts and vocabulary
needed to assess the emerging research
and clinical algorithms regarding char-
acterization of bacteria in the female
urinary tract.
Clinicians have a general awareness
of microbial communities (microbiota)
in diverse human anatomic sites (eg,
skin, mouth, bowel, and vagina). To
characterize many of these bacterial
communities, the National Institutes of
Health initiated the Human Microbiome
Project (HMP), which has spawned
overwhelming evidence that these
microbiota contribute to diverse human
health and disease states.
The terms
microbiome and microbiota often are
used interchangeably. In this article,
microbiota will refer to the microorgan-
isms that exist within a niche; micro-
biome will refer to the collection of all
their genomes.
From the Departments of Obstetrics & Gynecology and Urology (Dr Brubaker) and Department of Microbiology and Immunology (Dr Wolfe), Stritch School
of Medicine, Loyola University Chicago, Chicago, IL.
Received March 12, 2015; revised May 8, 2015; accepted May 17, 2015.
Loyola University Chicago Stritch School of Medicines research computing facility was developed through grant funds awarded by the Department of
Health and Human Services (1G20RR030939-01); our research has been supported by the Falk Foundation (LU#202567), by the National Institutes of
Health (R21DK097435-01A1, U10-HD054136), and by Astellas Medical and Scientic Affairs (Wolfe PI, VESI-12D01).
Dr Wolfe received an Investigator Initiated Grant from Astellas Medical and Scientic Affairs. Dr Brubaker reports no conict of interest.
Corresponding author: Linda Brubaker, MD, MS.
0002-9378 ª2015 The Authors. Published by Elsevier Inc. on behalf of ASCRS and ESCRS. This is an open access article under the CC BY-NC-ND license (http://
Emerging evidence challenges the long-held paradigm that the healthy bladder is sterile.
These discoveries may provide new opportunities to address important women’s health
conditions, which include preterm labor and delivery, urinary tract infections, and
common forms of urinary incontinence. Traditional tools for urinary bacterial assessment,
which includes urinary dipsticks and standard urine cultures, have significant limitations
that restrict the information that is available to clinicians. For example, the standard urine
culture does not detect slow-growing bacteria that die in the presence of oxygen. Two
new, complementary tools, however, can detect these and other organisms, which
permits a more complete characterization of bacterial communities within the female
bladder. Obstetrician-gynecologists should become familiar with these new approaches
(expanded quantitative urine culture and 16S ribosomal RNA gene sequencing) that can
detect previously unrecognized organisms. These advances are making it possible to
answer previously intractable scientific and clinical questions. Traditional nomenclature
used to describe the bacterial status in the bladder is quite dated and unsuited for the
emerging information about the bacterial milieu of the female urinary tract. In the context
of the sterile bladder paradigm, clinicians have learned about “uropathogens,”
“asymptomatic bacteriuria,” and “urinary tract infection.” Given that the lower urinary
tract is not sterile, these terms should be reevaluated. Clinicians can already benefit from
the emerging knowledge regarding urinary organisms that have previously gone un-
detected or unappreciated. For example, in some subpopulations of women with urinary
symptoms, existing data suggest that the urinary bacterial community may be associated
with women’s health conditions of interest. This Clinical Opinion highlights the in-
adequacies of the current tools for urinary bacterial assessment, describes the new
assessment tools, explains the current interpretation of the resulting data, and proposes
potential clinical uses and relevance. A new world is opening to our view that will give us
the opportunity to better understand urinary bacteria and the bladder in which they live.
This new knowledge has significant potential to improve patient care in obstetrics and
Key words: 16S rRNA sequencing, asymptomatic bacteriuria, microbiome, microbiota,
urgency urinary incontinence, urinary tract infection, urine culture
MONTH 2015 American Journal of Obstetrics &Gynecology 1
Clinical Opinion
The HMP and other studies of the
human microbiome typically identify
bacteria and eukaryotic microbes on the
basis of their DNA. Pioneered by mi-
crobial ecologists who needed a way to
identify organisms that could not be
cultured in the laboratory, these culture-
independent DNA-based approaches are
extremely powerful precisely because
they do not require isolation of
the bacterium. These DNA-based ap-
proaches generally take advantage of the
16S ribosomal RNA (rRNA) gene,
which encodes an essential component
of the ribosome. Because of this essen-
tiality, most of the 16S rRNA gene is
highly conserved. Between conserved
regions, however, some stretches of DNA
can evolve. The sequence differences in
these hypervariable regions serve as a
measure of evolutionary distance and
thus can be used to determine phyloge-
netic relatedness. All 9 known hyper-
variable regions (V1-V9) of the 16S
rRNA gene contain differences (called
polymorphisms) that can be used to
distinguish even closely related bacteria.
By comparing polymorphisms, re-
searchers can assign a DNA sequence
to the bacterium from which it
Many human niches contain massive
numbers of bacteria. For example, the
human colon contains 10
forming units (CFU) per gram of
To sequence large numbers of
genomes in large numbers of samples
simultaneously, researchers use multiple
massive parallel DNA-sequencing tech-
Also known as Next Genera-
tion sequencing, these revolutionary
technologies permit extremely high
throughput at low cost per base pair and
generate millions of sequence reads per
sample for multiple samples in a single
sequencing run. Sequencing technolo-
gies and techniques are advancing
rapidly with increased speed, availability,
and reliability at decreased cost. These
advances make it possible to answer
previously intractable scientic and
clinical questions. Further advances are
predicted to quickly move these tools
into the clinic.
Most clinicians are unfamiliar with
the attainment and presentation of DNA
sequencing data. The process begins the
moment the clinical sample is acquired.
To halt bacterial growth and maintain
DNA integrity, a preservative is added.
Next, the bacteria in the sample are
broken open, and the DNA is extracted.
With the use of the polymerase chain
reaction and universal primers, a hy-
pervariable region of the 16S rRNA gene
is amplied. To the resultant amplicons,
adaptor sequences are added. These se-
quences do 2 things. First, they contain
short stretches of DNA that adapt the
amplicons to the sequencing technology
of choice. Second, they contain distinct
DNA barcodes that permit multiplexing
(ie, simultaneous sequencing of ampli-
cons from multiple samples; as many as
384). This library of DNA fragments,
which represents the diversity of bacteria
present in the original samples, is now
sequenced. The output is thousands of
sequence readsthat are a digitized se-
ries of As, Gs, Cs, and Ts, that must be
processed. Then, the adaptor sequences
are removed, and the sequences are
demultiplexed and sorted by their barc-
odes into computerized bins, where each
bin represents the original sample from
which the read originated. The barcodes
are now removed, and the bioinformatic
analysis can begin. The intent is to assign
each processed read to a unique bacte-
rium, to compare the sequence of that
read to the 16S rRNA sequences of all
known bacteria. The resultant data are
often displayed as a histogram, wherein
each sample is represented as a bar and
each bacterium by a color (Figure 1).
Samples can be sorted on the basis of
their bacterial composition; the resultant
relationships are often represented by a
dendrogram (Figure 1). Sophisticated
bioinformatics and biostatistics ap-
proaches are then used to determine
associations with demographics, symp-
toms, and outcomes. This technology
will be used in many areas of clinical
medicine over the coming decade;
sequencing is already being incorporated
into urinary research.
The bladder was not included in the
initial HMP studies, presumably because
it was considered to be sterile. Existing
etiologic explanations and/or clinical
treatments for many common lower
urinary tract disorders are limited by this
long-held belief that the healthy female
urinary system contains no bacteria.
However, the DNA-based evidence sup-
porting the importance of microbiota in
other anatomic sites made it increasingly
implausible to think that the female
bladder would be entirely free of bac-
teria, especially given the bladders
anatomic location and its life events,
which include proximity to reproduc-
tive, sexual, and defecation functions.
Once sequencing was incorporated into
urinary research, multiple investigators
quickly conrmed that there is a resident
bacterial community in the urine (pre-
sumably from the bladder) of many
adult women.
Clearly, the lower
urinary tract is not sterile.
This important nding caused us to
reevaluate the reliability of the decades-
old approach to the standard clinical
urine culture. Most clinicians accept the
standard clinical urine culture as the
gold standard for bacterial testing in the
urine. The current broad interpretation
of the standard urine culture goes well
beyond the urine cultures very limited
initial role. In the 1950s, Edward
an infectious disease physician,
developed the standard urine culture,
using midstream urine and a cut-off of
CFU/mL to identify and prevent
post-operative sepsis in patients under-
going kidney surgery. Since Kasss orig-
inal work, other investigators have
attempted to rene the urine cul-
Most notably, Stamm et al
demonstrated that 10
of a known uro-
pathogen in the midstream urine of
women was indicative of lower UTI. In
hindsight, the evolving threshold for a
positiveurine culture may have been
an indication that there was more
complexity to the urinary bacterial
milieu in the bladder. The work of
Rosalind Maskell
is unknown to many;
yet, clearly ahead of her time, she per-
formed scientically rigorous studies
that provided compelling evidence to
disprove the dogma that urine was
sterile in the absence of a clinically rele-
vant infection. More recently, Hooten
et al
contributed evidence that the
bladder may also include many Gram-
positive bacteria, including lactobacilli,
Clinical Opinion Gynecology
2American Journal of Obstetrics &Gynecology MONTH 2015
staphylococci, streptococci, and Gard-
nerella vaginalis. They considered the
possibility that the bladder may have a
resident bacteria ora and suggested
reevaluation of the use of midstream
urine cultures for the diagnosis of lower
urinary tract symptoms. Given that the
clinicians have relied on the standard
urine culture test for decades, it is telling
that the debate about relevant thresholds
and specic organisms persists. What
accounts for our inability to nd
In the busy clinicians life, a urine
sample is collected and sent to a labo-
ratory, and a report is returned. What
goes on behind the scenes to produce
this report? Although there are differ-
ences across laboratories, the typical
standard urine culture protocol plates 1
L of urine on blood and MacConkey
agar and then incubates the sample at
35C in air for 24 hours. This protocol
was designed to detect a select group
of known uropathogens quickly, most
notably uropathogenic Escherichia coli
(UPEC), which causes most UTIs. This
protocol assumes that we know which
organisms are important to detect.
Although we clearly know some clini-
cally important organisms (such as
UPEC), the standard urine culture pro-
tocol was not designed to detect bacteria
that require special nutrients, grow
slowly, cannot tolerant oxygen, or are
present in small numbers (<10
mL). Some of these organisms may be
involved in urinary disorders. Moreover,
the assumption that urine is sterile has
led clinical microbiologists to set aside
bacterial colonies that resemble those
known to be part of the vaginal micro-
biota, because of a presumption that
lactobacillus and related organisms do
not live in the bladder. These limitations
of standard testing, which were designed
to detect a predetermined list of organ-
isms, block the ability to detect new or
previously unappreciated uropathogens.
Thus, clinicians get only a limited
glimpse of what is in their patientsurine
Clinicians have learned about uro-
pathogensas if we have a clearly
dened, complete list of such organisms.
In fact, there is an emerging group of
organisms that have previously gone
undetected or unappreciated. These or-
ganisms often require special culture
techniques, such as anaerobic condi-
tions. Ongoing research may reveal
organisms that have important in-
teractions with known uropathogens
(eg, UPEC) or independently may
cause human symptoms and/or disease.
Investigations into interactions with
well-studied uropathogens and their
virulence factors are beyond the scope of
this article.
Compared with the standard urine
culture, sequencing approaches provide
much more information about the or-
ganisms that are present in the urinary
microbiota. In women with and without
Relatedness of each microbiome profile
Measured by the Bray-Curtis method shown in a dendrogram (top) and by relative abundance of
classified sequences as shown in the histogram (bottom). In a dendrogram, the length of each
branch represents the relatedness between groups. The more related, the shorter the branch. We
can group each branch to determine clusters. The histogram displays the bacterial taxa that were
detected in each sample as a percent of the total sequence reads that were classified. Each color
represents a different family or genus. By comparing the dendrogram clustering to the classification,
we can define urotypes, which are named on the basis of the dominant (most common) classified
organism in each sample.
Brubaker. The female urinary microbiome. Am J Obstet Gynecol 2015. Gynecology Clinical Opinion
MONTH 2015 American Journal of Obstetrics &Gynecology 3
urinary symptoms, our research group
has compared the urinary microbiota
using standard urine culture and
sequencing approaches and found that
sequencing detects many more organ-
isms than does the standard urine cul-
A minority of urine samples
are sequence negativefor bacteria,
although it is our belief that these should
be considered a subthreshold for existing
technology, rather than lacking bacteria
altogether. Future studies will clarify
this distinction. Nonetheless, DNA
sequencing is clearly more sensitive
than a standard urine culture; indeed, it
may even be too sensitive for current
clinical use.
Beyond sensitivity concerns,
sequencing is not ready for front-line
clinical care for urinary testing. In
response to this clinical need, our
research group developed the expanded
quantitative urine culture (EQUC),
which goes beyond the duration and
conditions of the standard urine cul-
Using EQUC, we have shown that
the standard urine culture has an
astounding high false-negative rate, up
to 90%, depending on the clinical pop-
ulation of women without overt clinical
Khasriya et al
performed a
similar study and came to similar con-
clusions. These studies document the
inadequacy of current clinical culture-
based tests. To address the clinical
needs, our team is working to develop a
streamlined version of this technique
that could be performed in most, if not
all, clinical laboratory settings. However,
we stress that the simple presence of
bacteria in the urine should not be
equated with infection, nor should it
immediately prompt the use of systemic
antibiotics. It is quite possible that the
detected bacteria contribute to urinary
tract health.
This is a rapidly evolving scientic
landscape, with an increasing amount of
new evidence emerging as investigators
bring their expertise to questions that
will better inform clinical care. The
clinical community should be aware
of these 2 new tools, DNA sequencing
and EQUC, for urinary assessment
and be open to the possibilities that
previously undetected organisms may
play a role in certain womens
health conditions. Detailed descriptions
of these laboratory techniques are
What progress have we made with
these new tools? Using these 2 com-
plementary tools, our group and others
have provided compelling evidence that
most adult women have a resident
urinary microbiome, regardless of cur-
rent lower urinary tract symptoms.
We have found that these resident
urinary bacteria are clearly distinct
from bacteria that cause overt clinical
In some subpopulations of women
with urinary symptoms, we have
emerging evidence that the urinary
bacterial community may be associated
with a certain health status. For example,
in women who are affected by urgency
urinary incontinence (UUI), there is
some evidence that these communities
are associated with pretreatment UUI
symptoms and, perhaps, protection
against UTI.
Using EQUC, we showed
that the urinary microbiome of women
with UUI tends to be more diverse than
women without UUI.
Figure 2 pre-
sents these data as rarefaction curves,
which plot the accumulation of unique
species as participants are recruited and
analyzed. Rarefaction curves typically
are used to determine when a population
has been sampled fully and further
recruitment is not expected to identify
new species. Two curves that plateau at
different numbers of samples and/or at
different numbers of unique species are
evidence that the sampled populations
are distinct. This difference was mostly
due to a few species that are associated
strongly with the UUI cohort, including
Actinobaculum schaalii, Aerococcus uri-
nae, 2 Corynebacterium species, Lacto-
bacillus gasseri, Gardnerella vaginalis, and
Streptococcus anginosus. In contrast, L
crispatus was associated with controls.
These are exciting ndings that open
previously unappreciated opportunities
for scientic inquiry concerning pre-
vention, cause, and treatment of urinary
Rarefaction curves of cultured bacterial species by cohort
The cohorts were women with urgency urinary incontinence vs women without urgency urinary
incontinence. The plot depicts the number of unique species cultured via expanded quantitative urine
culture by the number of urines that were assayed.
Brubaker. The female urinary microbiome. Am J Obstet Gynecol 2015.
Clinical Opinion Gynecology
4American Journal of Obstetrics &Gynecology MONTH 2015
incontinence in women. Although the
current investigations have focused on
bacteria, other organisms could be pre-
sent, such as viruses or fungi.
The nomenclature used to describe
the bacterial status in the bladder is quite
dated and unsuited for the emerging
information about the female urinary
tract. For example, it is likely that the
dichotomous diagnosis of UTI will be
insufcient for clinical purposes. In
other parts of the human body, it is
recognized that resident bacterial com-
munities exist without infectionand
that infection can occur. In the urinary
tract, the threshold for infection,
formerly based on the standard urine
culture, will need to be reconsidered.
For example, asymptomatic bacteriuria
is a term used when the standard urine
culture detects a uropathogen above the
CFU/mL threshold of bacteria in an
individual with no lower urinary tract
symptoms. Obstetricians can appreciate
the importance of the detection of
a traditional group of uropathogens
because of the association with an
increased risk of preterm labor and de-
livery. Thus, the concept of asymptom-
atic bacteriuria is incorporated into
routine urinary screening, most often
with clinical dipsticks and perhaps
standard urine cultures. Yet, preterm
labor and delivery have proved to be
refractory to many treatment methods.
Given the current standard clinical
screening protocols focused on UTI
assessment during pregnancy, there is an
opportunity to apply these new tech-
niques to the detection of previously
unappreciated pathogens or urinary
microbiota characteristics of clinical
Asymptomatic bacteriuria is an espe-
cially challenging concept in the eld of
female pelvic medicine and reconstruc-
tive surgery. Using 16S rRNA sequencing
and EQUC, we have learned that most
women with UUI have a urinary
microbiota and that these women are
symptomatic; they typically have symp-
toms of urinary urgency, frequency, and
incontinence. Yet, their standard urine
culture is typically negative. Are these
women infectedor are these tradi-
tional clinical terms inadequate to
describe the clinical states that we are
now able to detect?
Simply put, the term asymptomatic
bacteriuria will become less and less
useful over time; science will be able to
better inform clinicians about the spe-
cic health condition of concern, rather
than our current nomenclature aggre-
gation of what may well be normal
with abnormalities of concern. A useful
concept for consideration is dysbiosis,
essentially an unhealthy perturbation in
the normal bacterial community of a
particular niche (eg, the bladder). As we
expand our understanding of the female
urinary microbiome, we will be able to
describe the normal range of urinary
microbiome states among groups of
women (for example, by age, hormonal
status, race, and ethnicity) and the clin-
ically relevant variations over time
within an individual woman.
We have entered a new era in our
understanding of the urinary bacterial
community in women. There is much to
learn! For years, we have cared for our
patients without this level of knowledge;
we now have an important opportunity
to improve patient care in obstetrics and
gynecology. Perhaps we have an oppor-
tunity to prevent certain conditions as
well. -
We thank the present and former members of
the Loyola Urinary Research and Education
Collaboration and acknowledge the Loyola
University Chicago Health Sciences Divisions
Ofce of Informatics and Systems Develop-
ment for their expertise and for the compu-
tational resources used in support of this
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Clinical Opinion Gynecology
6American Journal of Obstetrics &Gynecology MONTH 2015
... The existence of the urinary microbiome, the presence of bacterial communities within the urinary tract, is challenging the paradigm that this organ system is normally sterile (Siddiqui et al., 2012;Wolfe et al., 2012;Hilt et al., 2014;Brubaker and Wolfe, 2015;Thomas-White et al., 2016). Furthermore, several studies have shown an association between the urine microbiome and numerous urological diseases (Fouts et al., 2012;Siddiqui et al., 2012;Whiteside et al., 2015;Bajic et al., 2018;BucěvicṔ opovićet al., 2018;Magistro and Stief, 2019;Neugent et al., 2020). ...
... An alternative to urine cultures for detection of urinary microorganisms is polymerase chain reaction (PCR). PCR identifies organisms through the amplification of DNA material present in urine and many studies on the urinary microbiome rely on this molecular approach (Lewis et al., 2013;Brubaker and Wolfe, 2015;Ackerman et al., 2019). These methods do not discriminate between relic DNA (DNA from non-viable bacteria) versus DNA from viable bacteria. ...
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Background Polymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. Methods Varying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. Results PMA’s efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dC T =13.69) versus bacterial samples in unspun urine (dC T =1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups. Conclusion We have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.
... Recent investigations support the hypothesis that the bladder has its own local microbiome [4], a term that encompasses the combined genetic material of the microorganisms in this particular environment [5,6]. Due to new technologies, bacterial markers can be found in voided urine, which was previously thought to be sterile [7,8]. Since it has been shown that the human microbiota (i.e., the collection of microorganisms in a particular environment [9,10]) has a vast influence on the development of cancer (e.g., colorectal carcinoma, oropharyngeal carcinoma, and cervix carcinoma [7,8]), it seems reasonable to assume that this is also the case for the "Urobiome" (i.e., "microbial communities in the urinary tract" [11]) and the development of bladder cancer. ...
... Due to new technologies, bacterial markers can be found in voided urine, which was previously thought to be sterile [7,8]. Since it has been shown that the human microbiota (i.e., the collection of microorganisms in a particular environment [9,10]) has a vast influence on the development of cancer (e.g., colorectal carcinoma, oropharyngeal carcinoma, and cervix carcinoma [7,8]), it seems reasonable to assume that this is also the case for the "Urobiome" (i.e., "microbial communities in the urinary tract" [11]) and the development of bladder cancer. Nevertheless, the question remains to be answered whether the urinary or, in particular, the bladder microbiota is one of the missing pieces in the etiology of bladder cancer. ...
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The overall pathogenesis of bladder cancer is still unknown. The microbiota has been shown to play a critical role in the development of different types of cancer. Nevertheless, the role of the microbiota in the development of bladder cancer is still not fully discovered. This review aims to assess the urinary, vaginal, and intestinal microbiota analyzed from the bacterial, viral, and fungal compartments of bladder cancer patients compared with the microbiota of controls to reveal possible differences. A systematic review according to the PRISMA guidelines will be performed. The findings will be presented in narrative form as well as in tables and graphs.
... An urgent goal in urobiome research is to elucidate the mechanisms that link the urobiota to urinary conditions (1,8,9). This includes understanding why species can be associated with both asymptomatic and symptomatic states (65,66). ...
... In this study, we employed genomics to analyze the presence of plasmids in urinary isolates of 11 bacteria species relevant to urinary health. The contribution of plasmids to urinary health have not been thoroughly assessed, even in species in the families Enterobacteriaceae and Staphylococcaceae, which are arguably the best studied bacteria in microbiology (1,7,9). Plasmids have immense clinical and biological relevance, able to retain and transmit key traits like antibiotic resistance and virulence (15,20). ...
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The urinary tract has a microbial community (the urinary microbiota or urobiota) that has been associated with human health. Whole genome sequencing of bacteria is a powerful tool, allowing investigation of the genomic content of the urobiota, also called the urinary microbiome (urobiome). Bacterial plasmids are a significant component of the urobiome yet are understudied. Because plasmids can be vectors and reservoirs for clinically relevant traits, they are important for urobiota dynamics and thus may have relevance to urinary health. In this project, we sought plasmids in 11 clinically relevant urinary species: Aerococcus urinae, Corynebacterium amycolatum, Enterococcus faecalis, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Lactobacillus gasseri, Lactobacillus jensenii, Staphylococcus epidermidis, Streptococcus anginosus, and Streptococcus mitis. We found evidence of plasmids in E. faecalis, E. coli, K. pneumoniae, S. epidermidis, and S. anginosus but insufficient evidence in other species sequenced thus far. Some identified plasmidic assemblies were predicted to have putative virulence and/or antibiotic resistance genes, although the majority of their annotated coding regions were of unknown predicted function. In this study, we report on plasmids from urinary species as a first step to understanding the role of plasmids in the bacterial urobiota.
... In order to treat various diseases, modulation of microbial diversity by the investigation of prebiotics, probiotics or microbiota transplants as a therapeutic strategy is important [23]. Similarly, mapping the vaginal microbial communities in healthy females and recognizing changes in microbial configuration during vaginal disorders could revolutionize the way we treat vaginal disorders [24,25]. Furthermore, understanding the normal vaginal microbiota may alter our treatment approach, allowing us to place a greater emphasis on rebuilding a flexibly strong microbiome to reduce the host's inclination to bacteria rather than destroying pathogenic bacteria with antibiotics, which results in the impairment of healthy microbiota [26].The investigation of vaginal microbiota is a fast-growing discipline, i.e., in investigations of the optimal techniques for identification of microbial communities in the lower genital tract. ...
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Background: There is a unique microbial community in the female lower genital tract known as the vaginal microbiota, which varies in composition and density and provides significant benefits during pregnancy, reproductive cyclicity, healthy newborn delivery, protection from preterm birth, infections such as UTIs, bacterial vaginosis, and so on, and improves the efficacy of treatments for vaginal cancers. Methods: It is necessary to know how the vaginal microbiome is composed in order to make an accurate diagnosis of the diseases listed above. A microbiome’s members are difficult to classify, and the way microbial communities function and influence host–pathogen interactions are difficult to understand. More and more metagenomic studies are able to unravel such complexities due to advances in high-throughput sequencing and bioinformatics. When it comes to vaginal microbiota research, we’ll be looking at the use of modern techniques and strategies that can be used to investigate variations in vaginal microbiota in order to detect diseases earlier, better treat vaginal disorders, and boost women’s health. Discussion: The discussed techniques and strategies may improve the treatment of vaginal disorders and may be beneficial for women’s overall health.
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Recent advances in next-generation sequencing and metagenomic studies have provided insights into the microbial profile of different body sites. However, research on the microbial composition of urine is limited, particularly in children. The goal of this study was to optimize and develop reproducible metagenome and virome protocols using a small volume of urine samples collected from healthy children. We collected midstream urine specimens from 40 healthy children. Using the metagenomics shotgun approach, we tested various protocols. Different microbial roots such as Archaea, Bacteria, Eukaryota, and Viruses were successfully identified using our optimized urine protocol. Our data reflected much variation in the microbial fingerprints of children. Girls had significantly higher levels of Firmicutes, whereas boys had significantly higher levels of Actinobac-teria. The genus Anaerococcus dominated the urinary bacteriome of healthy girls, with a significant increase in Anaerococcus prevotii, Anaerococcus vaginalis, and Veillonella parvula (p-value < 0.001) when compared with that of boys. An increased relative abundance of Xylanimonas and Arthrobacter, with a significantly high abundance of Arthrobacter sp. FB24 (p-value 0.0028) and Arthrobacter aurescences (p-value 0.015), was observed in boys. The urinary mycobiome showed a significant rise in the genus Malassezia and Malassezia globose fungus (p-value 0.009) in girls, whereas genus Saccharomyces (p-value 0.009) was significantly high in boys. The beta diversity of the urinary mycobiome was found to differ between different age groups. Boys had significantly more Mastadenovirus and Human mas-tadenovirus-A in their urinary virome than girls. With increasing age, we noticed an increase in the relative abundance of the order Caudovirales. Our optimized protocols allowed us to identify the unique microbes for each sex by using an adequate volume of urine (3-10 mL) to screen for the bacteriome, mycobiome, and virome profiles in the urine of healthy children. To the best of our knowledge, this is the first study to characterize the metagenomics profiles of urine in a healthy pediatric population.
Introduction and hypothesis: We sought to evaluate patient satisfaction with a novel multiplex PCR UTI home collection kit for symptomatic UTI in a urogynecologic population. We secondarily sought to characterize reported uropathogens and resistance profiles of uropathogens in this population. We hypothesized that patients would be satisfied. Methods: This was a cross-sectional study of women who were surveyed later about their experience undergoing evaluation for a UTI with a home UTI test at a large tertiary care urogynecology practice in 2020. Symptomatic patients were sent a home UTI kit. We assessed patient satisfaction at a later time with a 5-point Likert scale and collected baseline information. The primary outcome was patient satisfaction with this experience. Secondary outcomes included type and number of uropathogens on testing. Results: A total of 30 patients [73% white race, mean age 71.9 (SD 12.0) years] were surveyed. Patients responded with a mean score of 4.7/5 to all satisfaction questions. Overall, 86% (26/30) of patients would choose this test again. Of those asked if they would choose this test again outside of the COVID-19 pandemic, 86% responded affirmatively. The most common symptoms reported included dysuria (53%), urgency (37%) and frequency (30%). The most common pathogens identified included Escherichia coli (70%), Enterococcus faecalis (60%) and Aerococcus urinae (43%). Conclusions: Patients were satisfied with home UTI PCR testing and the majority would choose this option again. Home UTI PCR testing revealed common uropathogens for a population with a high proportion of recurrent UTI, but additional research comparing home versus in-office urine PCR testing is necessary.
Contrary to dogma, the human urinary bladder possesses its own unique bacterial community with alteration of composition associated with disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease often characterized by kidney impairment.
Stress urinary incontinence is common in pregnancy and its severity can be reduced with pelvic floor muscle training. Overactive bladder syndrome prevalence increases with gestation and treatment can be conservative or medical. Pelvic organ prolapse is multifactorial. Pelvic floor exercises and pessaries are important treatments and previous surgical management can affect the mode of delivery. Recurrent urinary tract infections can be treated with antibiotic prophylaxis or with non‐antibiotic prophylaxis such as methanamine hippurate, D‐mannose and hygiene behaviour. Urinary retention can occur at any point during pregnancy, causing bladder distension, voiding dysfunction and subsequent lifelong catheterisation. To be aware of the pathophysiology and management of different types of urinary tract dysfunction in pregnancy and the postpartum period. To understand the potential causes and management of pelvic organ prolapse in pregnancy and the postpartum period. To understand the different potential management options in recurrent lower urinary tract infections.
The discovery of microbial communities in the urinary tract (the urobiome) has fundamentally altered the previous doctrine regarding urine sterility and associated urinary disorders. Recent advances in culturing and culture-independent DNA sequencing technologies have characterised the resident microbial community in the urobiome, and has, in turn, demonstrated how community imbalances potentially contribute to infection and disease. As we enter a post-antibiotic era, the effectiveness of standard antimicrobial treatments against multi-drug resistant (MDR) uropathogens is vastly diminished. Preliminary research is accumulating surrounding microbiome-based therapies, and their potential as non-antibiotic therapeutics. In this context, the urobiome is significantly underexplored, and knowledge regarding the fundamental role of its constituents is lacking. Herein, we review the current state of the art concerning the urobiome; specifically, how it impacts health and disease states, in the context of urinary tract infections (UTIs). Furthermore, we discuss the development of novel biological therapeutics that may have the potential to provide significant advancements in UTI therapy, with particular focus on bacterial interference, probiotics, antimicrobial peptides, bacteriocins, and bacteriophage.
Improvements in bacterial culturing and DNA sequencing techniques have revealed a diverse, and hitherto unknown, urinary tract microbiome (urobiome). The potential role of this microbial community in contributing to health and disease, particularly in the context of urinary tract infections (UTIs) is of significant clinical importance. However, while several studies have confirmed the existence of a core urobiome, the role of its constituent microbes is not yet fully understood, particularly in the context of health and disease. Herein, we review the current state of the art, concluding that the urobiome represents an important component of the body's innate immune defences, and a potentially rich resource for the development of alternative treatment and control strategies for UTIs.
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Resident bacterial communities (microbiota) and host antimicrobial peptides (AMPs) are both essential components of normal host innate immune responses that limit infection and pathogen induced inflammation. However, their interdependence has not been investigated in the context of urinary tract infection (UTI) susceptibility. Here, we explored the interrelationship between the urinary microbiota and host AMP responses as mechanisms for UTI risk. Using prospectively collected day of surgery (DOS) urine specimens from female pelvic floor surgery participants, we report that the relative abundance and/or frequency of specific urinary microbiota distinguished between participants who did or did not develop a post-operative UTI. Furthermore, UTI risk significantly correlated with both specific urinary microbiota and β-defensin AMP levels. Finally, urinary AMP hydrophobicity and protease activity were greater in participants who developed UTI, and correlated positively with both UTI risk and pelvic floor symptoms. These data demonstrate an interdependency between the urinary microbiota, AMP responses and symptoms, and identify a potential mechanism for UTI risk. Assessment of bacterial microbiota and host innate immune AMP responses in parallel may identify increased risk of UTI in certain populations.
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Culture-independent methods in microbiology (quantitative PCR (qPCR), sequencing, microarrays, direct from sample matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS), etc.) are disruptive technology. Rather than providing the same results as culture-based methods more quickly, more cheaply or with improved accuracy, they reveal an unexpected diversity of microbes and illuminate dark corners of undiagnosed disease. At times, they overturn existing definitions of presumably well-understood infections, generating new requirements for clinical diagnosis, surveillance and epidemiology. However, current diagnostic microbiology, infection control and epidemiology rest principally on culture methods elegantly optimized by clinical laboratorians. The clinical significance is interwoven; the new methods are out of context, difficult to interpret and impossible to act upon. Culture-independent diagnostics and surveillance methods will not be deployed unless the reported results can be used to select specific therapeutics or infection control measures. To cut the knots surrounding the adoption of culture-independent methods in medical microbiology, culture-dependent methods should be supported by consistent culture-independent methods providing the microbial context. This will temper existing biases and motivate appropriate scrutiny of the older methods and results.
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Bacterial DNA and live bacteria have been detected in human urine in the absence of clinical infection, challenging the prevailing dogma that urine is normally sterile. Urgency urinary incontinence (UUI) is a poorly understood urinary condition characterized by symptoms that overlap urinary infection, including urinary urgency and increased frequency with urinary incontinence. The recent discovery of the urinary microbiome warrants investigation into whether bacteria contribute to UUI. In this study, we used 16S rRNA gene sequencing to classify bacterial DNA and expanded quantitative urine culture (EQUC) techniques to isolate live bacteria in urine collected by using a transurethral catheter from women with UUI and, in comparison, a cohort without UUI. For these cohorts, we demonstrated that the UUI and non-UUI urinary microbiomes differ by group based on both sequence and culture evidences. Compared to the non-UUI microbiome, sequencing experiments revealed that the UUI microbiome was composed of increased Gardnerella and decreased Lactobacillus. Nine genera (Actinobaculum, Actinomyces, Aerococcus, Arthrobacter, Corynebacterium, Gardnerella, Oligella, Staphylococcus, and Streptococcus) were more frequently cultured from the UUI cohort. Although Lactobacillus was isolated from both cohorts, distinctions existed at the species level, with Lactobacillus gasseri detected more frequently in the UUI cohort and Lactobacillus crispatus most frequently detected in controls. Combined, these data suggest that potentially important differences exist in the urinary microbiomes of women with and without UUI, which have strong implications in prevention, diagnosis, or treatment of UUI.
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This study's aims were to detect and quantify bacterial DNA in the urine of randomized trial participants about to undergo treatment for urinary urgency incontinence (UUI) without clinical evidence of urinary tract infection (UTI) and to determine if the presence of bacterial DNA in baseline urine relates to either baseline urinary symptoms or UTI risk after urinary tract instrumentation. Women without clinical evidence of baseline UTI were randomized to cystoscopic onabotulinum toxin A injection and oral placebo medication versus cystoscopic placebo injection and active oral medication. Bacterial DNA in participants' catheterized urine was measured by quantitative polymerase chain reaction (qPCR). Bacterial DNA was detected in the urine of 38.7 % of participants (60 out of 155). In these 60 qPCR-positive participants, baseline daily UUI episodes were greater than in the 95 qPCR-negative participants (5.71 [±2.60] vs 4.72 [±2.86], p = 0.004). Neither symptom severity by questionnaire nor treatment outcome was associated with qPCR status or with qPCR level in qPCR-positive subjects. In contrast, the presence of urinary bacterial DNA was associated with UTI risk: only 10 % of the qPCR-positive women developed a UTI post-treatment, while 24 % of the qPCR-negative women did so. The median qPCR level for qPCR-positive samples did not differ significantly by UTI status (UTI 2.58 × 10(5) vs no UTI 1.35 × 10(5) copies/mL, p = 0.6). These results may indicate a urinary bacterial contribution to both baseline UUI and the risk of post-treatment UTI.
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The polyphasic approach used today in the taxonomy and systematics of the Bacteria and Archaea includes the use of phenotypic, chemotaxonomic and genotypic data. The use of 16S rRNA gene sequence data has revolutionized our understanding of the microbial world and led to a rapid increase in the number of descriptions of novel taxa, especially at the species level. It has allowed in many cases for the demarcation of taxa into distinct species, but its limitations in a number of groups have resulted in the continued use of DNA-DNA hybridization. As technology has improved, next-generation sequencing (NGS) has provided a rapid and cost-effective approach to obtaining whole-genome sequences of microbial strains. Although some 12 000 bacterial or archaeal genome sequences are available for comparison, only 1725 of these are of actual type strains, limiting the use of genomic data in comparative taxonomic studies when there are nearly 11 000 type strains. Efforts to obtain complete genome sequences of all type strains are critical to the future of microbial systematics. The incorporation of genomics into the taxonomy and systematics of the Bacteria and Archaea coupled with computational advances will boost the credibility of taxonomy in the genomic era. This special issue of International Journal of Systematic and Evolutionary Microbiology contains both original research and review articles covering the use of genomic sequence data in microbial taxonomy and systematics. It includes contributions on specific taxa as well as outlines of approaches for incorporating genomics into new strain isolation to new taxon description workflows.
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Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative using standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these [48/52 (92%)] were reported as no growth at 10(3) colony forming units (CFU)/mL by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%) and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.
A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.
Background: The cause of acute uncomplicated cystitis is determined on the basis of cultures of voided midstream urine, but few data guide the interpretation of such results, especially when gram-positive bacteria grow. Methods: Women from 18 to 49 years of age with symptoms of cystitis provided specimens of midstream urine, after which we collected urine by means of a urethral catheter for culture (catheter urine). We compared microbial species and colony counts in the paired specimens. The primary outcome was a comparison of positive predictive values and negative predictive values of organisms grown in midstream urine, with the presence or absence of the organism in catheter urine used as the reference. Results: The analysis of 236 episodes of cystitis in 226 women yielded 202 paired specimens of midstream urine and catheter urine that could be evaluated. Cultures were positive for uropathogens in 142 catheter specimens (70%), 4 of which had more than one uropathogen, and in 157 midstream specimens (78%). The presence of Escherichia coli in midstream urine was highly predictive of bladder bacteriuria even at very low counts, with a positive predictive value of 10(2) colony-forming units (CFU) per milliliter of 93% (Spearman's r=0.944). In contrast, in midstream urine, enterococci (in 10% of cultures) and group B streptococci (in 12% of cultures) were not predictive of bladder bacteriuria at any colony count (Spearman's r=0.322 for enterococci and 0.272 for group B streptococci). Among 41 episodes in which enterococcus, group B streptococci, or both were found in midstream urine, E. coli grew from catheter urine cultures in 61%. Conclusions: Cultures of voided midstream urine in healthy premenopausal women with acute uncomplicated cystitis accurately showed evidence of bladder E. coli but not of enterococci or group B streptococci, which are often isolated with E. coli but appear to rarely cause cystitis by themselves. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.).