Review TRENDS in Microbiology
9 September 2004
Intracellular bacterial communities of
uropathogenic Escherichia coli in
urinary tract pathogenesis
Gregory G. Anderson1, Karen W. Dodson1, Thomas M. Hooton2 and Scott J. Hultgren1
1
Department of Molecular Microbiology, Washington University School of Medicine, Box 8230, 660 S. Euclid Ave., St Louis, MO
63110, USA
2
Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington School of Medicine, Box 359930,
Harborview Medical Center, 325 Ninth Ave., 2WC-516, Seattle, WA 98104, USA
Urinary tract infections in young, healthy women
frequently recur, despite their traditional classification
as acute infections. Conventional wisdom dictates that
uropathogens causing recurrent infections in such
individuals come from the fecal or vaginal flora, in the
same manner as the initial infection. However, recent
studies of uropathogenic Escherichia coli have found
that it can carry out a complex developmental program
within the superficial epithelial cells of the mouse
bladder, forming intracellular bacterial communities
with many biofilm-like properties. These intracellular
biofilms allow the bacteria to outlast a strong host
immune response to establish a dormant reservoir of
pathogens inside the bladder cells. Re-emergence of
bacteria from this reservoir might be the source
of recurrent infection.
Urinary tract infections (UTIs), considered among the
most common of bacterial diseases, afflict a large pro-
portion of the world population [1]. A majority of these
infections occur in young, healthy women. In a large
prospective study of young, sexually active women, the
incidence of UTI was w0.5 per person-year [2]. More
recently, a population-based survey estimated that as
many as 11 million women in the United States had at
least one presumed UTI treated with an antibiotic in 1995,
and that the cost for evaluation and management of UTIs
was 1.6 billion dollars [1]. In this survey, the self-reported
incidence of UTI in women 18 years and older was 10.8%,
and the cumulative lifetime risk of UTI was 60%. Even
episodes of acute, uncomplicated UTI are associated with
considerable morbidity, including 6.1 days of symptoms,
2.4 days of restricted activity and 0.4 bed days [1]. Given
that an estimated 130–175 million cases of UTI occur
annually worldwide [3], the societal costs associated with
UTI are huge. The most common isolates from patients are
uropathogenic Escherichia coli (UPEC), accounting for
w80% of all acute, community-acquired UTIs [4]. Aside
from UPEC, several other microorganisms can also cause
UTI, most notably Staphylococcus saprophyticus, impli-
cated in most of the remaining UTI cases [5]. Patients with
Corresponding author: Scott J. Hultgren (hultgren@borcim.wustl.edu).
Available online 24 July 2004
www.sciencedirect.com 0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.07.005
Vol.12 No.
complicating factors, such as diabetes, spinal cord inju-
ries, urinary catheters, or hospitalization, can also present
with UTI caused by Klebsiella pneumoniae, Proteus
mirabilis, Enterococcus faecalis, Pseudomonas aerugi-
nosa, Serratia marcescens and group B streptococci [4].
Although traditionally thought of as acute and self-
limiting, 27–44% of women with an initial UTI will
experience at least one recurrence of symptoms within
six months, despite antibiotic therapy [5,6]. Although
these recurrent infections might occasionally be due to a
persistent focus of infection, the majority have been
thought to be reinfections caused by the initially infecting
strain persisting in the fecal flora [7]. The bacteria
associated with recurrent UTI often appear to be pheno-
typically or genetically identical to the bacterial strain
that caused the initial infection, suggesting that selected
E. coli strains might become uniquely adapted for
colonizing and infecting their respective hosts [7]. The
steady increase of antibiotic resistance [8] and occurrences
of clonal outbreaks of UPEC-associated UTIs [9] highlight
the need for greater understanding of the mechanisms of
UPEC pathogenesis.
UPEC have proven to be quite stealthy in its patho-
genesis of the urinary tract, using a complex pathway to
mediate infection and persistence in the face of a strong
innate host immune response. One of the most studied
abilities of UPEC is binding host tissues and particular
organ niches. UPEC accomplish this binding by assem-
bling several different adhesive organelles on their sur-
face, the most understood comprising P pili and type 1 pili
[10]. These pili, assembled by the chaperone–usher path-
way, display an adhesin molecule at their distal end [11].
P pili display the PapG adhesin, which has been shown to
be required for pyelonephritic pathogenesis by binding to
globoside present on human kidney cells [12]. By contrast,
type 1 pili have been found to be vital for attachment to the
bladder epithelium. The FimH adhesin on type 1 pili binds
mannose, and in the bladder FimH recognizes mannosy-
lated residues present on uroplakin proteins that line the
luminal surface of the bladder superficial epithelial cells
[13]. FimH-mediated binding of the bladder epithelium is
the initial step in an intricate cascade of events leading to
 Review TRENDS in Microbiology Vol.12 No.9 September 2004 425

the myriad of symptoms associated with acute UTI and systems, including aerobactin [24] and the more recently
possible long-term residence of UPEC in the urinary tract. described IroN system [25,26]. These iron siderophores
scavenge available iron and enhance UPEC survival in the
nutrient-limiting bladder environment. Finally, most
UPEC virulence determinants
UPEC strains produce an acidic polysaccharide capsule,
Other than type 1 and P pili, UPEC produce additional
which protects the bacteria from phagocytosis by human
factors that influence disease progression (Table 1). Some
PMNs and inhibits activation of complement [24].
of these virulence determinants are located on one of
several UPEC specific pathogenicity-associated islands
Host response to UPEC infection
[14]. A recent genomic analysis of a UPEC strain revealed
The host counters the actions of UPEC upon the bladder
the presence of genes for ten putative chaperone–usher
by mounting a robust and dramatic response. Bladder
pilus systems, two putative type IV pili and at least seven
superficial epithelial cells express Toll-like receptor 4
putative autotransporter proteins [15]. The large reper-
(TLR-4) on their membrane, which, along with CD14,
toire of pilus systems might confer upon UPEC multiple
recognizes lipopolysaccharide from the bacteria and
binding specificities and the capacity to colonize various
activates an innate immune response [27,28]. A burst of
sites throughout the urinary tract and other environ-
inflammatory cytokines leads to a massive infiltration of
ments. Autotransporter proteins can also have adhesive
neutrophils to fight the infection [29,30]. Further,
properties or they might fill other roles, such as toxins,
increased transcription of inducible nitric oxide synthase
proteases, invasins, serum resistance factors and motility
by PMNs and the bladder epithelium results in high levels
mediators [16]. One UPEC specific autotransporter, Sat,
of nitric oxide and related breakdown products, which
appears to exert a toxic effect upon urinary tract cells
could have toxic effects on the bacteria [31,32]. Upon
in vitro [17]. Incubation of bladder and kidney cells with
infection, the bladder epithelium is triggered to exfoliate
Sat-producing UPEC leads to extreme vacuolation in the
superficial facet cells [33]. This aids clearance of any
cytoplasm of the host cells and possible loosening of
tissue-associated bacteria [34]. The underlying epi-
cellular junctions. In a mouse model of UTI, Sat also
thelium, normally extremely inert [35], rapidly undergoes
induces cytoplasmic vacuolation and severe histological
a renewal process wherein underlying epithelial cells
damage in kidneys of infected laboratory animals [18].
proliferate and differentiate to replace the superficial
Two other UPEC-expressed autotransporter proteins, Pic
layer of cells [36]. Despite the defensive immune arsenal
and Tsh, appear to have serine protease activity [19].
and rapid epithelial turnover, UPEC are able to colonize
These two proteins are expressed during infection in
the bladder and establish a persistent reservoir which can
laboratory mice and appear to be generally associated
last at least several months after infection in laboratory
with pyelonephritis strains. UPEC also express an RTX
mice [37]. Bacteria persist in the bladder notwithstanding
(repeat in toxin) toxin called a-hemolysin (HlyA), often
antibiotic treatment [38], and, periodically, bacteria
linked with the P pilus operon [20]. HlyA forms pores in a
arising from this reservoir are shed in the urine of these
variety of host cell membranes and can induce calcium
mice. Human patients have also been shown to shed
oscillations in proximal tubule cells of infected rat kidneys
bacteria in the urine, even in the absence of active UTI
[21]. These fluctuations might serve as second messengers
symptoms [39,40]. Therefore, UPEC appear to be quite
during immune activation of the host. Another toxin
adept at establishing a protective niche for themselves in
produced by UPEC, cytotoxic necrotizing factor 1 (CNF-1),
the bladder and persisting for extended periods of time.
influences the host cell cytoskeleton by targeting the Rho
family of GTP-binding proteins [22]. CNF-1 has been
Intracellular bacterial communities
shown to kill human bladder epithelial cells in vitro and
Recently, it was discovered that UPEC activate a complex
inhibit phagocytosis of bacteria by human polymorpho-
developmental cascade upon their entry into superficial
nuclear leukocytes (PMNs) [23]. In addition to proteases
bladder cells [41,42]. This cascade was elucidated using
and toxins, UPEC produce several iron acquisition
state of the art microscopy, including scanning and
Table 1. Virulence determinants of uropathogenic Escherichia transmission electron microscopy, immunohistochemistry
coli and time-lapse videomicroscopy. For videomicroscopy,
mouse bladders infected with green fluorescence protein-
Factor Refs expressing UPEC were stretched on an incubation
Adhesins P pili [10] chamber and placed on an epifluorescence microscope
Type 1 pili [10] [42]. Images were captured every 30 seconds to two
S pili [10]
Dr adhesins [10]
minutes, and multiple overlapping time frames were
Toxins HlyA [20] examined to piece together a timeline of UTI progression.
CNF-1 [23] Using these techniques, it was found that, in the early
Sat [18] stages, the bacteria invade the superficial cells and rapidly
Siderophores Aerobactin [24] divide. As the bacteria grow, dramatic phenotypic
IroN [26]
IreA [24]
switches result in the establishment of intracellular
Proteases Pic [19] bacterial communities (IBCs) (Figure 1). IBCs progress
Tsh [19] through several stages, culminating in the formation of
Other Capsule [24] biofilm-like communities inside the superficial cells.
LPS [24]
Eventually, bacteria detach from the biofilm and burst
www.sciencedirect.com
426 Review TRENDS in Microbiology
Figure 1. Middle intracellular bacterial communities (IBCs) are seen as ‘pods’ on the
surface of infected mouse bladder epithelium. (a) Numerous pods cover the
bladder surface in this scanning electron micrograph. Formation of biofilm-like
pods protects the bacteria from immune clearance, thus allowing uropathogenic
Escherichia coli (UPEC) to build up to extremely high levels in the bladder. Scale
bar, 50 mm. (b) Magnification of a pod reveals a smooth shell which is the uroplakin-
coated luminal membrane of the bladder superficial epithelial cell. The pod consists
of one cell that is completely filled with intracellular bacteria. Scale bar, 5 mm. (c) An
IBC is visible inside the bladder epithelium by fluorescent microscopy. Scale bar,
20 mm. The green fluorescence protien-expressing bacteria form a dense sphere
within the bladder cell. IBC progression can be monitored by time-lapse
videomicroscopy, in which time-lapse images are captured by epifluorescence
microscopy [42].
out into the bladder lumen. These escaped bacteria then
rebind to the epithelium and initiate another round of IBC
formation. The downstream effects of this cascade might
permit UPEC to evade the host immune response and
persist in the urinary tract.
Binding and early IBC formation
Type 1 pili, with the FimH adhesin at the distal tip, are
crucial for UPEC attachment to the bladder epithelium
[43]. Two lines of evidence confirm the vital role of FimH
for bacterial attachment to the epithelium. First, isogenic
fimH mutants are defective for binding and colonization of
the bladder [33]. Second, polystyrene latex beads coated
with FimH readily associate with, and are internalized by,
human bladder epithelial cells in vitro [44]. FimH binds to
mannosylated uroplakins on the bladder epithelium via a
deep acidic pocket formed at the tip of the lectin-binding
domain [13]. Four different uroplakin molecules associate
into hexameric rings, which are arrayed in crystalline
plaques on the luminal membrane of the bladder super-
ficial epithelial cells. This uroplakin plaque provides a
permeability barrier to prevent toxic molecules concen-
trated in the urine from diffusing into deeper tissues [35,45].
High resolution freeze-fracture electron microscopy has
revealed that the tips of type 1 pili, which contain the
adhesin, are buried in the central cavity of the uroplakin
hexamers, tethering the bacteria to the epithelium [33]. It
was recently shown that shear force enhances FimH-
mediated binding [46], and such forces when encountered
www.sciencedirect.com
Vol.12 No.9 September 2004
in the bladder might increase the ability of type 1 piliated
organisms to attach and colonize the urinary tract.
After bacterial attachment, UPEC quickly invade the
bound epithelial cell [44,47]. This key event occurs one to
three hours after initial inoculation. It has been shown
using tissue culture models that binding activates the Rho
family of small GTP-binding proteins, specifically RhoA,
Cdc42 and Rac1, which then activate focal adhesin kinase,
phosphoinositide 3-kinase, a-actinin and vinculin. These
processes result in localized actin rearrangements and
membrane extensions around the bacteria [47]. The
membrane engulfs the bacteria, leading to UPEC intern-
alization via zipper-like phagocytosis.
Once intracellular, UPEC rapidly grow and divide,
forming small clusters of bacteria, termed ‘early IBCs’
[42]. Early IBCs have previously been called ‘bacterial
factories’ owing to the ability of UPEC to usurp the
superficial bladder cells and convert them into factories
for bacterial growth [37]. A major benefit that invasion
and establishment of an intracellular community confers
upon UPEC is protection from killing by antibiotics, which
has been shown both in vitro and in vivo [37,38]. In this
way, UPEC form a protective niche in the bladder where
they can hide and survive. The bacteria within the early
IBC divide rapidly during the first six to eight hours after
inoculation, with a doubling time of w30 to 35 minutes.
The bacteria at this stage orient themselves randomly in
the cytoplasm of the host cell, such that they form an
amorphous, loosely associated bacterial clump [42].
During this period of quick expansion, UPEC maintain
their typical rod morphology, with an average length of
3 mm. This fast growth leads to a surge of microorganisms
within the bladder early in infection.
IBC maturation and middle IBCs
Between six to eight hours postinoculation, early IBCs
experience a dramatic phenotypic switch [42]. A remark-
able drop in bacterial growth rate, resulting in doubling
times greater than 60 minutes, takes place concurrent
with a significant shortening of bacterial morphology to an
average 0.7 mm. At the same time, the amorphous clump
appears to come together to form a tight, highly organized
sphere inside the host cell. These events represent a
maturation of the early IBC into a middle IBC stage with
many biofilm-like properties [41].
Bacterial biofilms are often associated with long-term
persistence of organisms in various environments. Bio-
films are collections of bacteria that are attached to a
surface, or to each other, and that display community
behavior [48]. Initial events in biofilm formation involve
binding of planktonic bacteria to a surface and formation
of small bacterial clusters called microcolonies. As these
microcolonies grow into mature biofilms, the bacteria
differentiate, undergo dramatic phenotypic changes and
encase themselves in an extracellular polysaccharide
matrix [49,50]. These actions result in a large three-
dimensional structure, with subpopulations of bacteria at
different regions of this structure displaying altered gene
transcriptional patterns [48]. It is thought that these
subpopulations represent a division of labor, where each
group performs certain specialized functions that benefit
 Review TRENDS in Microbiology
the whole community [51]. As the biofilm ages, a subset of
bacteria detach from the biofilm and revert to a planktonic
state, which allows for spread of organisms to other niches
[48]. A biofilm lifestyle benefits the bacteria by providing
protection from environmental insults and changes [49].
Bacteria in biofilms display dramatically increased resist-
ance to antibiotics because of the inability of antibiotics to
penetrate the biofilm matrix, the slow growth-rate of the
bacteria in the biofilm, physiological changes in the
bacteria and biofilm-state expression of factors that
directly inhibit antibiotic activity [48,52]. In addition,
biofilms confer increased resistance to host immune effects
upon their constituent bacteria by concealing the bacteria
inside the biofilm matrix. For instance, bacteria in
biofilms are protected from opsonizing antibodies [48].
Phenotypic variation and differentiation might also
enhance bacterial resistance to PMNs and immune
effector molecules [48]. Antibiotic and immune resistances
facilitate bacterial biofilm survival in vivo during many
disease conditions, including native valve endocarditis,
otitis media, chronic bacterial prostatitis, periodontitis
and cystic fibrosis. Bacterial biofilms have also been
described on inert medical devices, such as prosthetic
heart valves, central venous catheters, contact lenses,
intrauterine devices, urinary catheters and dental unit
water lines [48]. The presence of biofilms in disease or on
medical devices leads to chronic symptoms, owing to
persistence of bacteria, despite antimicrobial therapy.
An important factor in E. coli biofilm formation is type 1
pili, which aids stable attachment of the bacteria to a
surface [53]. Flagella also assist biofilm formation by
mediating initial weak surface contact and influencing
expansion of biofilms across a surface [53]. Antigen 43, an
outer membrane autotransporter protein, promotes auto-
aggregation of E. coli and can assist interbacterial
interactions during microcolony formation [54]. This
might also be the function of secreted amyloid fibers
called curli [55,56]. The regulator of the curli operon,
CsgD, might also regulate several metabolic and other
factors that influence formation of mature biofilms [57].
A key process during biofilm maturation, following
attachment and microcolony formation, is secretion of
colanic acid, an exopolysaccharide that creates a poly-
saccharide matrix consisting of a complex three-dimen-
sional architecture [58].
The incredible organization and structure produced by
UPEC in the middle IBC are highly reminiscent of
bacterial biofilms [49]. As middle IBCs grow, the bacterial
mass pushes against the epithelial cell membrane,
creating a podlike protrusion on the surface of infected
bladder epithelium (Figure 1) [41]. Pods correspond to a
middle IBC. The bacteria in the middle IBC, although free
in the cytoplasm, stay together as a spherical community,
filling up much of the intracellular volume of a superficial
umbrella cell. The surface of the pod is the luminal
membrane of the bladder cell, which is stretched tight,
appearing smooth by scanning electron microscopy, and is
coated with an impermeable shell of uroplakin. The
bacteria inside the pod are highly organized and sur-
rounded by numerous fibers emanating from the bacterial
surface. These fibers make extensive contacts with the
www.sciencedirect.com
Vol.12 No.9 September 2004 427
surrounding matrix, creating individualized compart-
ments for each bacterium that establish a spatial
orientation of microorganisms inside the IBC. UPEC
express several factors in the middle IBC which are
important for in vitro biofilm formation, including type
1 pili and antigen 43 [41]. Similar to biofilms,
expression of these factors is heterogeneous, with the
bacteria setting up subpopulations with differential
gene expression. Polysaccharides are also present
throughout the middle IBC.
By differentiating into an intracellular biofilm, UPEC
construct a safe haven where they can hide and wait out
the tide of the host immune attack. PMNs recruited to the
bladder are targeted to the IBCs and attempt to clear the
bacteria [29,42]. However, the IBC remains largely
unaffected. The uroplakin shell covering the IBC appears
to protect UPEC by denying the PMNs access to the
bacteria. The structural organization of the pod might also
protect the bacteria from antibiotic susceptibility. The
matrix surrounding the bacteria inside the cell might also
add an additional layer of protection from antibiotics and
the immune system. It is interesting that in vitro biofilm-
state UPEC have been found to be resistant, even when
antibiotics reach the bacteria [59], suggesting that the
physiological state of the microbes in biofilms enhances
bacterial survival. In this manner, UPEC are able to
replicate to large numbers, unperturbed by the host and
other insults.
Late IBCs and fluxing
Eventually, the IBC progresses to a late stage [42].
Starting around 12 hours postinoculation, bacteria on
the edge of the intracellular biofilm detach from the group
and differentiate into a typical rod morphology, with an
average length of 2 mm. At this stage of the maturation
cycle, the bacteria become highly motile. These motile
bacteria swim toward the edge of the cell, burst out into
the bladder lumen in a process termed ‘fluxing’ and spread
out over the epithelium. Fluxing bacteria have also been
observed erupting en masse from the epithelial cell, the
entire community simultaneously emerging from its
intracellular hiding place [37]. It is possible that fluxing
initiates with a few bacteria, and as the host cell
membrane integrity decreases, some signal leads to the
concomitant bursting of the entire IBC. From the volume
of mature IBCs and the size of the bacteria, it is estimated
that an average IBC contains in the order of 105
organisms. Fluxing of multiple IBCs, therefore, might
result in the release of hundreds of thousands or millions
of UPEC back into the bladder lumen, leading to spread
within the bladder as well as dissemination back into the
environment [42]. Escaped bacteria often filament, reach-
ing up to 70 mm or greater in length [37,42]. Although the
mechanisms and purpose behind filamentation remain
unclear, PMNs appear to have little effect upon the
filaments [42]. This allows UPEC to survive long enough
in the inflamed bladder to reattach to the epithelium.
UPEC persistence in the bladder
A second round of IBC formation occurs as escaped
bacteria rebind and reinvade the superficial cells [42].
428 Review TRENDS in Microbiology Vol.12 No.9 September 2004

Binding and invasion Early IBC Middle IBC Late IBC

TRENDS in Microbiology

Figure 2. Schematic representation of the intracellular bacterial community (IBC) maturation program of uropathogenic Escherichia coli (UPEC) within the superficial
epithelium of the mouse bladder. Soon after inoculation, UPEC (green) bind and invade the superficial epithelial cells of the bladder in a process mediated by type 1 pili (red).
Rapid multiplication leads to formation of a disorganized bacterial clump within the cell cytoplasm, known as an ‘early IBC’. Activation of developmental cascades results in
maturation to a biofilm-like middle IBC. An impermeable uroplakin shell and the biofilm-like environment protect UPEC in the middle IBC from immune attack. Eventually,
UPEC detach from the intracellular biofilm, become motile (yellow flagella) and flux out of the cell into the bladder lumen. This presumably facilitates bacterial persistence
within the host and transmission to the environment. Fluxed UPEC can rebind to the epithelium to form additional generations of IBCs. The time scale for IBC progression is
dependent upon the bacterial strain and the host strain used. During infection with UPEC strain UTI89 in C3H/HeN mice, the first round of IBC development generally
completes within 24 hours after inoculation [42]. After several days, a quiescent bacterial reservoir develops in the bladder, which could be a seed for urinary tract infection
recurrence. Reproduced, with permission, from Ref. [42].

However, this second round takes much longer to
progress, with doubling times in the early IBC of greater
than 60 minutes. Multiple IBC cycles probably occur but
after several days, only a quiescent reservoir of small
intracellular bacterial clusters (doublets, quadruplets,
small groups) remains in the bladder. The bladder
epithelium is intact at this stage, with the bacteria
residing inside superficial cells which appear smaller
than normal. These smaller superficial cells might result
from growth and differentiation of the underlying epi-
thelial cells following exfoliation of the original superficial
cells. As infection progresses, a greater number of fluxed
UPEC might bind and invade the underlying epithelium,
exposed during exfoliation. Physiological differences
between the superficial and underlying cells influence
whether IBCs form. Indeed, IBCs have never been seen
within the underlying bladder cells. During this reservoir
stage, no apparent actions are taken either by the host or
the bacteria, and UPEC can persist in this state in the
bladder for months after an initial infection [37,38].
Periodically during this persistent stretch, large numbers
of organisms are seen in the urine [38]. It is thought that
reactivation of UPEC from their dormant reservoir state,
and subsequent multiplication inside the bladder epi-
thelium to re-form IBCs, lead to this bacterial spike
in the urine.
transmission back into the environment, subsequent IBC
cycles and establishment of a chronic bacterial reservoir.
Alternatively, formation of the reservoir might occur
through an entirely separate route. Continuing studies
will provide answers to this and other fundamental
questions relating to UPEC pathogenesis and persistence
(Box 1). The elucidation of the IBC program was carried
out using a mouse model of infection. However, UPEC also
bind, invade and replicate inside human epithelial cells
in vitro [37,44,47], and it will be vital for future studies to
establish whether IBCs and quiescent reservoirs of
bacteria form in human patients. As more is understood
about the factors that are expressed during UTI, and their
regulation, it will be essential to determine their role in
the IBC program. Such knowledge will provide a compre-
hensive understanding of UTI progression and lead to new
clues about how to abrogate UPEC infection. Further,
because many different microorganisms invade host
tissues and multiply inside host cells, it is possible that
an IBC-like developmental program is important during
various stages of other chronic recurrent infections.
A greater understanding of the nature of intracellular
bacterial communities in chronic or recurrent infections
will aid the development of new and more effective
treatments for these problematic diseases.

Box 1. Intriguing questions for future study

Clinical impact
The discovery of the IBC maturation cascade suggests
that UPEC possess a program that enables the bacteria to
evade early innate immune defenses. Considering that
extracellular bacteria are readily cleared by PMNs [42],
the formation of intracellular communities of bacteria
inside the superficial cells of the bladder appears key to
the survival of UPEC in the urinary tract during the acute
phases of infection (Figure 2). IBCs appear to permit
UPEC to survive the immune onslaught that occurs at
these early stages. In this haven, the bacteria can safely
multiply, creating an overpowering legion which then
escapes into the bladder lumen. Presumably, the sheer
numbers of bacteria that escape overwhelm the immune
system, leading to several potential downstream effects:
www.sciencedirect.com
† Do intracellular bacterial communities (IBCs) form in human
patients with cystitis?
† What genetic mechanisms do uropathogenic Escherichia coli
(UPEC) use to form IBCs?
† What accounts for polymorphonuclear leukocyte resistance by the
filaments?
† Why do IBCs form only in the superficial epithelial cells of the
bladder?
† What induces UPEC to become quiescent?
† Can stimulation of the dormant reservoir lead to a urinary tract
infection recurrence?
† What signals activate the dormant reservoir?
† How and when do known virulence determinants influence the IBC
cascade?
† What bacterial and/or host signals regulate IBC processes?
† How do IBCs relate to the chronic phase of infection?
† Are IBCs formed in other chronic recurrent infections?
 Review TRENDS in Microbiology
Acknowledgements
We thank S. Justice, C. Hung and J. Palermo for helpful discussions. This
work was supported by National Institutes of Health grants AI29549,
DK51406 and AI48689, and by ORWH SCOR grant DK64540 in
partnership with DHHS, NIDDK and the FDA.
References
1 Foxman, B. (2002) Epidemiology of urinary tract infections: incidence,
morbidity, and economic costs. Am. J. Med. 113(Suppl. 1), 5S–13S
2 Hooton, T.M. et al. (1996) A prospective study of risk factors for
symptomatic urinary tract infection in young women. N. Engl. J. Med.
335, 468–474
3 Russo, T.A. and Johnson, J.R. (2003) Medical and economic impact of
extraintestinal infections due to Escherichia coli: focus on an
increasingly important endemic problem. Microbes Infect. 5, 449–456
4 Ronald, A. (2002) The etiology of urinary tract infection: traditional
and emerging pathogens. Am. J. Med. 113(Suppl. 1), 14S–19S
5 Hooton, T.M. and Stamm, W.E. (1997) Diagnosis and treatment of
uncomplicated urinary tract infection. Infect. Dis. Clin. North Am. 11,
551–581
6 Ikaheimo, R. et al. (1996) Recurrence of urinary tract infection in a
primary care setting: analysis of a 1-year follow-up of 179 women.
Clin. Infect. Dis. 22, 91–99
7 Stapleton, A. and Stamm, W.E. (1997) Prevention of urinary tract
infection. Infect. Dis. Clin. North Am. 11, 719–733
8 Stamm, W.E. (2002) Scientific and clinical challenges in the manage-
ment of urinary tract infections. Am. J. Med. 113(Suppl. 1), 1S–4S
9 Manges, A.R. et al. (2001) Widespread distribution of urinary tract
infections caused by a multidrug-resistant Escherichia coli clonal
group. N. Engl. J. Med. 345, 1007–1013
10 Mulvey, M.A. (2002) Adhesion and entry of uropathogenic Escherichia
coli. Cell. Microbiol. 4, 257–271
11 Sauer, F.G. et al. (2000) Chaperone-assisted pilus assembly and
bacterial attachment. Curr. Opin. Struct. Biol. 10, 548–556
12 Dodson, K.W. et al. (2001) Structural basis of the interaction of the
pyelonephritic E. coli adhesin to its human kidney receptor. Cell 105,
733–743
13 Hung, C.S. et al. (2002) Structural basis of tropism of Escherichia
coli to the bladder during urinary tract infection. Mol. Microbiol.
44, 903–915
14 Hacker, J. et al. (1999) Pathogenicity islands of extraintestinal
Escherichia coli. In Pathogenicity Islands and Other Mobile Virulence
Elements (Kaper, J.B. and Hacker, J. eds), pp. 59–75, American
Society for Microbiology
15 Welch, R.A. et al. (2002) Extensive mosaic structure revealed by the
complete genome sequence of uropathogenic Escherichia coli. Proc.
Natl. Acad. Sci. U. S. A. 99, 17020–17024
16 Henderson, I.R. and Nataro, J.P. (2001) Virulence functions of
autotransporter proteins. Infect. Immun. 69, 1231–1243
17 Guyer, D.M. et al. (2000) Identification of sat, an autotransporter toxin
produced by uropathogenic Escherichia coli. Mol. Microbiol. 38, 53–66
18 Guyer, D.M. et al. (2002) Sat, the secreted autotransporter toxin of
uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder
and kidney epithelial cells. Infect. Immun. 70, 4539–4546
19 Heimer, S.R. et al. (2004) Autotransporter genes pic and tsh are
associated with Escherichia coli strains that cause acute pyelone-
phritis and are expressed during urinary tract infection. Infect.
Immun. 72, 593–597
20 Stanley, P. et al. (1998) Acylation of Escherichia coli hemolysin: a
unique protein lipidation mechanism underlying toxin function.
Microbiol. Mol. Biol. Rev. 62, 309–333
21 Uhlen, P. et al. (2000) Alpha-haemolysin of uropathogenic E. coli
induces Ca2C oscillations in renal epithelial cells. Nature 405,
694–697
22 Mills, M. et al. (2000) Cytotoxic necrotizing factor type 1 of
uropathogenic Escherichia coli kills cultured human uroepithelial
5637 cells by an apoptotic mechanism. Infect. Immun. 68, 5869–5880
23 Hofman, P. et al. (2000) Escherichia coli cytotoxic necrotizing factor-1
(CNF-1) increases the adherence to epithelia and the oxidative burst
of human polymorphonuclear leukocytes but decreases bacteria
phagocytosis. J. Leukoc. Biol. 68, 522–528
www.sciencedirect.com
Vol.12 No.9 September 2004 429
24 Johnson, J.R. (2003) Microbial virulence determinants and the
pathogenesis of urinary tract infection. Infect. Dis. Clin. North Am.
17, 261–278
25 Russo, T.A. et al. (1999) Identification of genes in an extraintestinal
isolate of Escherichia coli with increased expression after exposure to
human urine. Infect. Immun. 67, 5306–5314
26 Sorsa, L.J. et al. (2003) Characterization of an iroBCDEN gene cluster
on a transmissible plasmid of uropathogenic Escherichia coli:
evidence for horizontal transfer of a chromosomal virulence factor.
Infect. Immun. 71, 3285–3293
27 Schilling, J.D. et al. (2003) Toll-like receptor 4 on stromal and
hematopoietic cells mediates innate resistance to uropathogenic
Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 100, 4203–4208
28 Schilling, J.D. et al. (2003) CD14- and Toll-like receptor-dependent
activation of bladder epithelial cells by lipopolysaccharide and type 1
piliated Escherichia coli. Infect. Immun. 71, 1470–1480
29 Schilling, J.D. et al. (2001) Bacterial invasion augments epithelial
cytokine responses to Escherichia coli through a lipopolysaccharide-
dependent mechanism. J. Immunol. 166, 1148–1155
30 Haraoka, M. et al. (1999) Neutrophil recruitment and resistance to
urinary tract infection. J. Infect. Dis. 180, 1220–1229
31 Poljakovic, M. and Persson, K. (2003) Urinary tract infection in iNOS-
deficient mice with focus on bacterial sensitivity to nitric oxide. Am.
J. Physiol. Renal Physiol. 284, F22–F31
32 Poljakovic, M. et al. (2001) Escherichia coli-induced inducible nitric
oxide synthase and cyclooxygenase expression in the mouse bladder
and kidney. Kidney Int. 59, 893–904
33 Mulvey, M.A. et al. (2000) Bad bugs and beleaguered bladders:
interplay between uropathogenic Escherichia coli and innate host
defenses. Proc. Natl. Acad. Sci. U. S. A. 97, 8829–8835
34 Gunther, N.W.T. et al. (2001) In vivo dynamics of type 1 fimbria
regulation in uropathogenic Escherichia coli during experimental
urinary tract infection. Infect. Immun. 69, 2838–2846
35 Southgate, J. et al. (1999) Urothelial tissue regulation. Unraveling the
role of the stroma. Adv. Exp. Med. Biol. 462, 19–30
36 Mysorekar, I.U. et al. (2002) Molecular regulation of urothelial
renewal and host defenses during infection with uropathogenic
Escherichia coli. J. Biol. Chem. 277, 7412–7419
37 Mulvey, M.A. et al. (2001) Establishment of a persistent Escherichia
coli reservoir during the acute phase of a bladder infection. Infect.
Immun. 69, 4572–4579
38 Schilling, J.D. et al. (2002) Effect of trimethoprim-sulfamethoxazole
on recurrent bacteriuria and bacterial persistence in mice infected
with uropathogenic Escherichia coli. Infect. Immun. 70, 7042–7049
39 Anderson, M. et al. (2004) Viable but nonculturable bacteria are
present in mouse and human urine specimens. J. Clin. Microbiol. 42,
753–758
40 Hooton, T.M. et al. (2000) A prospective study of asymptomatic
bacteriuria in sexually active young women. N. Engl. J. Med. 343,
992–997
41 Anderson, G.G. et al. (2003) Intracellular bacterial biofilm-like pods in
urinary tract infections. Science 301, 105–107
42 Justice, S.S. et al. (2004) Differentiation and developmental pathways
of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc.
Natl. Acad. Sci. U. S. A. 101, 1333–1338
43 Gunther, I.N. et al. (2002) Assessment of virulence of uropathogenic
Escherichia coli type 1 fimbrial mutants in which the invertible
element is phase-locked on or off. Infect. Immun. 70, 3344–3354
44 Martinez, J.J. et al. (2000) Type 1 pilus-mediated bacterial invasion of
bladder epithelial cells. EMBO J. 19, 2803–2812
45 Lewis, S.A. (2000) Everything you wanted to know about the bladder
epithelium but were afraid to ask. Am. J. Physiol. Renal Physiol. 278,
F867–F874
46 Thomas, W.E. et al. (2002) Bacterial adhesion to target cells enhanced
by shear force. Cell 109, 913–923
47 Martinez, J.J. and Hultgren, S.J. (2002) Requirement of Rho-family
GTPases in the invasion of Type 1-piliated uropathogenic Escherichia
coli. Cell. Microbiol. 4, 19–28
48 Donlan, R.M. and Costerton, J.W. (2002) Biofilms: survival mechan-
isms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15,
167–193
49 Dunne, W.M., Jr. (2002) Bacterial adhesion: seen any good biofilms
lately? Clin. Microbiol. Rev. 15, 155–166
430 Review TRENDS in Microbiology Vol.12 No.9 September 2004

50 O’Toole, G. et al. (2000) Biofilm formation as microbial development.
Annu. Rev. Microbiol. 54, 49–79
51 Webb, J.S. et al. (2003) Bacterial biofilms: prokaryotic adventures in
multicellularity. Curr. Opin. Microbiol. 6, 578–585
52 Mah, T.F. et al. (2003) A genetic basis for Pseudomonas aeruginosa
biofilm antibiotic resistance. Nature 426, 306–310
53 Pratt, L.A. and Kolter, R. (1998) Genetic analysis of Escherichia coli
biofilm formation: roles of flagella, motility, chemotaxis and type I pili.
Mol. Microbiol. 30, 285–293
54 Danese, P.N. et al. (2000) The outer membrane protein, antigen 43,
mediates cell-to-cell interactions within Escherichia coli biofilms. Mol.
Microbiol. 37, 424–432
55 Prigent-Combaret, C. et al. (2001) Complex regulatory network
controls initial adhesion and biofilm formation in Escherichia coli
via regulation of the csgD gene. J. Bacteriol. 183, 7213–7223
56 Chapman, M.R. et al. (2002) Role of Escherichia coli curli operons in
directing amyloid fiber formation. Science 295, 851–855
57 Brombacher, E. et al. (2003) The curli biosynthesis regulator CsgD
co-ordinates the expression of both positive and negative determi-
nants for biofilm formation in Escherichia coli. Microbiol. 149,
2847–2857
58 Danese, P.N. et al. (2000) Exopolysaccharide production is required for
development of Escherichia coli K-12 biofilm architecture.
J. Bacteriol. 182, 3593–3596
59 Stone, G. et al. (2002) Tetracycline rapidly reaches all the constituent
cells of uropathogenic Escherichia coli biofilms. Antimicrob. Agents
Chemother. 46, 2458–2461

Have you contributed to an Elsevier publication?

Did you know that you are entitled to a 30% discount on books?
A 30% discount is available to ALL Elsevier book and journal contributors when ordering books or stand-alone CD-ROMs directly
from us.
To take advantage of your discount:
1. Choose your book(s) from www.elsevier.com or www.books.elsevier.com
2. Place your order
Americas:
TEL: +1 800 782 4927 for US customers
TEL: +1 800 460 3110 for Canada, South & Central America customers
FAX: +1 314 453 4898
E-MAIL: author.contributor@elsevier.com
All other countries:
TEL: +44 1865 474 010
FAX: +44 1865 474 011
E-MAIL: directorders@elsevier.com
You’ll need to provide the name of the Elsevier book or journal to which you have contributed. Shipping is FREE on pre-paid
orders within the US, Canada, and the UK.
If you are faxing your order, please enclose a copy of this page.
3. Make your payment
This discount is only available on prepaid orders. Please note that this offer does not apply to multi-volume reference works or
Elsevier Health Sciences products.

www.books.elsevier.com

www.sciencedirect.com