Molecular Immunology 108 (2019) 56–67

Contents lists available at ScienceDirect

Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Urinary tract infection: Pathogenicity, antibiotic resistance and T

development of effective vaccines against Uropathogenic Escherichia coli
Mohammad Reza Asadi Karam, Mehri Habibi , Saeid Bouzari
⁎ ⁎⁎

Department of Molecular Biology, Pasteur Institute of Iran, Pasteur Ave., Tehran, 13164, Iran

ARTICLE INFO ABSTRACT

Keywords: Urinary tract infections (UTIs) are recognized as one of the most common infectious diseases in the world that
Urinary tract infection can be divided to different types. Uropathogenic Escherichia coli (UPEC) strains are the most prevalent causative
Uropathogenic Escherichia coli agent of UTIs that applied different virulence factors such as fimbriae, capsule, iron scavenger receptors, flagella,
Virulence factors toxins, and lipopolysaccharide for their pathogenicity in the urinary tract. Despite the high pathogenicity of
Immune responses
UPEC strains, host utilizes different immune systems such as innate and adaptive immunity for eradication of
Antibiotic resistance
Vaccine
them from the urinary tract. The routine therapy of UTIs is based on the use of antibiotics such as β-lactams,
trimethoprim, nitrofurantoin and quinolones in many countries. Unfortunately, the widespread and misuse of
these antibiotics resulted in the increasing rate of resistance to them in the societies. Increasing antibiotic re-
sistance and their side effects on human body show the need to develop alternative strategies such as vaccine
against UTIs. Developing a vaccine against UTI pathogens will have an important role in reduction the mortality
rate as well as reducing economic costs. Different vaccines based on the whole cells (killed or live-attenuated
vaccines) and antigens (subunits, toxins and conjugatedvaccines) have been evaluated against UTIs pathogens.
Furthermore, other therapeutic strategies such as the use of probiotics and antimicrobial peptides are considered
against UTIs. Despite the extensive efforts, limited success has been achieved and more studies are needed to
reach an alternative of antibiotics for treatment of UTIs.

1. Urinary tract infection
1.1. Definition of urinary tract infection
The urinary tract infection (UTI) occurs when the pathogen is able
to enter the urinary tract system and reach more than 105 colony/ml in
urine (Smelov et al., 2016). UTI is known as the second common cause
of infectious diseases (Klumpp et al., 2006). According to the previous
studies, UTI accounts for approximately 40% of all infections acquired
in the hospitals and 50% of bacteremia that can prolong the hospita-
lization and increase the morbidity and mortality rate of patients
(Mathai et al., 2001; Saint et al., 2008). In the United States, about 11
million people causing to UTI have been annually referred to the
healthcare centers, and approximately 470,000 have been hospitalized,
which annually costs was about $6 billion (Mann et al., 2017;
Subashchandrabose and Mobley, 2015). In general, it is estimated that
nearly half of women and 12% of men will experience at least one UTI
during their lifetimes, and a quarter of these people will have the re-
currence form of disease in the future (Brumbaugh et al., 2013).
1.2. Classification of urinary tract infections
Urinary tract infections have the ability to develop disease in var-
ious types, including asymptomatic bacteriuria, acute, chronic, and
recurrent infection (Smelov et al., 2016). The incidence of three or
more UTIs per year, as well as 2 or more UTIs in less than 6 months is
considered as the recurrent UTI, which are the major challenge in
treatment of UTI patients (Nuutinen and Uhari, 2001; Terlizzi et al.,

Abbreviations: UTI, urinary tract infection; UPEC, Uropathogenic Escherichia coli; E. coli, Escherichia coli; IBC, Intracellular bacterial communities; LPS,
Lipopolysaccharide; DAF, Decay-accelerating factor; RBC, Red Blood Cell; HlyA, alpha-hemolysin; CNF1, Cytotoxic necrotizing factor 1; Ag43, Antigen 43; Vat,
Vacuolating autotransporter cytotoxin (Vat); Sat, Secreted autotransporter toxin; TLR, Toll like receptor; NK, Natural killer; PAMP, Pathogen-Associated Molecular
Pattern; IFN-γ, Interferon gamma; CLSI, Clinical and Laboratory Standards Institute; Mar, Multiple antibiotic resistance locus; ESBL, Extended Spectrum β-Lactamase;
PMQR, plasmid-mediated quinolone resistance; CT, Cholera toxin; MPL, Monophosphoryl Lipid A
⁎
Corresponding author at: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran. No. 69, Pasteur Ave, Tehran, 1316943551, Iran.
⁎⁎
Corresponding author.
E-mail addresses: m_habibi@pasteur.ac.ir (M. Habibi), bouzari@pasteur.ac.ir (S. Bouzari).

https://doi.org/10.1016/j.molimm.2019.02.007
Received 4 August 2018; Received in revised form 2 February 2019; Accepted 12 February 2019
0161-5890/ © 2019 Elsevier Ltd. All rights reserved.
M.R. Asadi Karam, et al.
2017). Acute UTIs can be classified into two categories, lower UTIs
(cystitis) and upper UTIs (pyelonephritis) (Foxman, 2014; Smelov et al.,
2016). Because some of the symptoms of cystitis and pyelonephritis are
similar, identification the type of infection (lower or upper UTIs) should
be performed on the basis of clinical examinations and the laboratory
results (Foxman, 2014; Mulvey, 2002). UTIs can also clinically be
classified into two types, complicated and uncomplicated UTIs (Zacche
and Giarenis, 2016). Complicated UTIs are attributed to the infection in
patients with impaired or obstructed urinary tract system, or patients
that use the medical devices such as catheters, which their treatment
sometimes requires more challenges for physicians. Uncomplicated UTI
is commonly found in patients with a healthy urinary tract system and
without using the medical devices, which is often seen in outpatients
(community-acquired infections) (Hooton, 2012; Lichtenberger and
Hooton, 2008; Mann et al., 2017).
1.3. Causative agents of urinary tract infection
Most UTIs are caused by bacteria, which the most common of them
belonged to the Enterobacteriaceaefamily (Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, Citrobacter and Enterobacter),
Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus,
Staphylococcus saprophyticus, Streptococcus and Enterococcus faecalis
(Flores-Mireles et al., 2015; Mann et al., 2017).
Uropathogenic Escherichia coli (UPEC) are a heterogeneous group of
extra intestinal pathogenic E. coli (ExPEC) that seem to originate within
the gut and can be disseminated via oral-faecal routes, contaminated
food products, and sexual contact (Dhakal et al., 2008). To date, seven
major phylogroups of E. coli have been identified (A, B1, B2, C, D, E and
F) based on the phenotypic and genotypic properties that UPEC strains
have shown to belong mainly to phylogenetic groups B2 and D
(Clermont et al., 2013; Ejrnaes, 2011). UPEC strains as the most pre-
valent cause of UTIs account for about 80% of uncomplicated UTIs,
95% of community-acquired infections, and also the half of hospital-
acquired infections (Dhakal et al., 2008; Foxman, 2010; Kucheria et al.,
2005). In the various studies that have been conducted by our research
team on the uropathogens collected from inpatients and outpatients, it
was found that UPEC isolates were the most common isolate in the UTI
patients (Tabasi et al., 2015, 2016).
2. Virulence properties of UPEC strains
2.1. The pathogenesis process of UPEC strains
Studies have shown that UPEC strains are able to develop UTI in a
complex process. After entering the intestinal E. coli strain with the
origin of normal flora from the urethra into the urinary tract system, it
reaches the bladder and binds to the surface epithelium of bladder
using the bacterial adhesin factors (Mann et al., 2017). These bacteria
can be internalized into the facet cells of bladder and changed the
circumstances for their survival. Of course, the host immune system will
also apply its strategies to exclude these internalized bacteria. In the
next step, the bacteria can enter the cytoplasm of the bladder cells and
after replication, form components called intracellular bacterial com-
munities (IBCs) (Wright et al., 2007). The host immune system ex-
foliates some of the IBCs by shedding the bladder surface facet cells into
the urine (McLellan and Hunstad, 2016; Mulvey et al., 1998). The re-
mained structures are then able to convert into biofilm-like structures
that are resistant to antibiotics and immune responses (Anderson et al.,
2003; Schwartz et al., 2011). Finally, some of these bacteria escape the
biofilm structures and convert to the filamentous and motile form, in
order to disseminate into the bladder lumen (Flores-Mireles et al., 2015;
Justice et al., 2004). This form of bacteria has the ability of binding and
invasion to naive bladder cells and re-formation of IBCs (Spaulding and
Hultgren, 2016). It seems that some of the IBC forms remain inactive in
the bladder cells and as resistant reservoirs are able to re-activate and
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Molecular Immunology 108 (2019) 56–67
cause recurrent UTIs (McLellan and Hunstad, 2016; Wiles et al., 2008).
In addition to these intracellular reservoirs, recurrent UTIs may be
caused by the re-entering of E. coli strains from the gut into the urinary
tract system which require further studies.
2.2. Virulence factors of UPEC strains
UPEC strains encode a group of virulence factors which are essential
for their pathogenicity and survival in the urinary tract. The previous
studies have demonstrated that mutations in these factors can cause
functional defects in the pathogenicity of these strains (Weichhart et al.,
2008). These factors include a variety of adhesins, iron scavenger re-
ceptors, secreted toxins, capsule, flagella, outer membrane proteins and
lipopolysaccharide (LPS) that are transferred by genetic elements such
as plasmids, transposons, bacteriophages, and pathogenicity islands
(Bien et al., 2012; Terlizzi et al., 2017).
2.2.1. Fimbrial adhesins
Two groups of pili including mannose-sensitive and mannose-re-
sistant pili have been identified in UPEC strains (Schembri et al., 2001).
Type 1 pili are seemed to be the most important mannose-sensitive pili
in these strains, which are encoded by a gene cluster in the bacterial
chromosome. The large component of the pili has been made from a
heterodimer, called FimA, and the small components that include FimC,
FimD, FimF, FimH and FimG (Schembri et al., 2001; Thankavel et al.,
1997). FimH adhesin at the tip of the pili probably is the main factor in
the binding of UPEC to the mannosylated uroplakins on the surface of
the bladder epithelial cells. The binding of bacteria through the FimH
adhesin stimulates the entry of bacteria into the bladder epithelial cells
and the exfoliation or apoptosis of the epithelial cells, which these
properties suggest the role of type 1 pili in formation of IBCs, cell in-
vasion, and the formation of resistant biofilm-like structures (Dhakal
et al., 2008; Eto et al., 2007).
Mannose-resistant pili are highly diverse in UPEC strains, which
divided into different types, based on the receptor characteristics and
the other properties. P pili are one of the most known fimbriae that are
made from repeating units of PapA and at the tip of this structure there
is an adhesin called PapG, which is equivalent to FimH in type 1 pili,
and is communicated with three other subunits called PapE, PapF and
PapK (Busch and Waksman, 2012; Kline et al., 2009; Mulvey, 2002).
Studies have shown that P pili play an important role in colonization of
UPEC in the kidneys (Roberts et al., 1994), which has been demon-
strated in studies done by our research team (Tabasi et al., 2015, 2016).
S pili, as other mannose-resistant pili, are also made from a major
subunit called SfaA plus three subunits, called SfaG, SfaH and SfaS,
which the SfaS is the adhesin of the pili and causes the binding of
bacteria to sialic acid structures on the kidney epithelial cells (Hanisch
et al., 1993; Mulvey, 2002). The pili are often seen in E. coli strains
which are the cause of more severe infections such as pyelonephritis,
meningitis and bacteremia (Parkkinen et al., 1988). The F1C pili are
also an analogue of S pili, which unlike S pili its specific receptor is
glycolipid receptors harboring β-galactosidase on the bladder and
kidney epithelial cells (Khan et al., 2000). Another group of mannose-
resistant pili is Dr family that is able to bind human Decay-accelerating
factor (DAF) molecules on red blood cells (RBCs) and epithelial cells,
which this binding to kidneys facilitates colonization and prolongs
bacterial survival in the urinary tract. Interestingly, people infected
with UPEC strains harboring Dr adhesin showed a higher risk of causing
to recurrent UTIs (Mulvey, 2002; Nowicki et al., 2001).
2.2.2. Nonfimbrial (afimbrial) adhesins
TosA, as a putative repeat-in-toxin (RTX) family member and a
known nonfimbrial adhesin showed a role in colonization of UPEC in
the urinary tract of animal model (Vigil et al., 2011). The members of
autotransporter family such as FdeC (for factor adherence E. coli), An-
tigen-43 (Ag43), UpaH, UpaC and UpaG also were defined as other
M.R. Asadi Karam, et al.
important nonfimbrial adhesins among the UPEC strains (Allsopp et al.,
2012b; Nesta et al., 2012; Wells et al., 2010). As a surface factor, Ag43
showed different roles in pathogenicity of UPEC strains such as binding,
colonization, formation of IBCs and biofilm, and long-term stability of
bacteria in the bladder (Ulett et al., 2007). The UpaG trimeric auto-
transporter is also involved in binding of bacteria to bladder epithelial
cells and extracellular matrix proteins such as fibronectin as well as cell
aggregation and biofilm formation (Valle et al., 2008). In several stu-
dies, contribution of UpaH in biofilm formation and colonization of the
mice bladder was shown in in vitro and in vivo conditions (Allsopp et al.,
2012a, 2010). It is mentioned that further studies about the im-
munogenicity and protection efficacy of UpaH as a vaccine candidate
against clinical UPEC isolates are ongoing by our research team. Among
the other nonfimbrial adhesins, an iron-regulating adhesin known as
Iha (IrgA homologue adhesin) is also shown to have a role in the
binding of bacteria to the bladder epithelial cells (Johnson et al., 2005).
2.2.3. Iron scavenger receptors
UPEC strains utilize multiple strategies to absorb iron from the ur-
inary tract of host. One of these systems is the use of iron chelators
called siderophores to adsorb iron and bring it into the cytosol of
bacteria (Reigstad et al., 2007). These siderophores in UPEC strains
include the salmochelin, aerobactin, enterobactin, and yersiniabactin
that their presence has been shown in studies performed by us and
other researchers (Caza and Kronstad, 2013; Garcia et al., 2011; Habibi
et al., 2017). In addition, iron receptors Hma and ChuA produced by
UPEC strains are important proteins that uptake the heme and transfer
it into the periplasm of UPEC (O’Brien et al., 2016; Reigstad et al.,
2007). Studies showed that outer membrane iron receptors have also
other important roles for UPEC such as colonization, biofilm production
and formation of IBC reservoirs (Flores-Mireles et al., 2015).
2.2.4. Toxins
In contrast to the diarrheagenic E. coli, UPEC strains lack the type III
secretion system and use the type I and V secretion systems to secrete
toxins (Henderson et al., 2004). These toxins are including alpha-he-
molysin (hlyA), Cytotoxic necrotizing factor 1 (CNF1), and auto-
transporter toxins such as Vacuolating autotransporter cytotoxin (Vat),
and Secreted autotransporter toxin (Sat) (Wiles et al., 2008). These
toxins cause defects in the function or shape of the host cells, stop the
cell cycle, or lysis cells (Wiles et al., 2008). Alpha-hemolysin is a cal-
cium-dependent secretion protein produced by half of the UPEC strains,
and has the ability of lysis the bladder and kidney cells, invasion, sti-
mulation of cytokines release, and induction of the inflammatory re-
sponses (Marrs et al., 2005; Nielubowicz and Mobley, 2010). CNF1
toxin has been observed in approximately 30% of UPEC strains, and led
to the constitutive activation of the Rho family from GTP-binding
proteins, including Cdc42, which results in cytoskeleton disruption in
the host cells. This toxin also plays a role in the binding and invasion of
UPEC to the host cells (Lemonnier et al., 2007; Tabasi et al., 2016). Sat
and Vat autotransportertoxins as a type of serine proteases showed le-
thal effects on the bladder and kidney cells in in vitro and caused tissue
damages especially glomerular damage and vacuolization of kidneys in
mouse model (Guyer et al., 2002; Maroncle et al., 2006; Wiles et al.,
2008).
2.2.5. Flagella (Flagellum)
Bacterial flagella are composed of a basal body, hook, motor, and
filament (Honko and Mizel, 2005). The flagella are made from protein
monomers called flagellin (FliC) which by activation of Toll like re-
ceptor-5 (TLR5) have been used in vaccine purposes (Honko and Mizel,
2005; Salazar-Gonzalez and McSorley, 2005). In UPEC strains, fla-
gellum is used to reach the new nutrients, and escape from unfavorable
conditions and the host immune system (Lane et al., 2007). Different
studies have shown that the expression of flagella in UPEC strains is
coincident with ascension of the bacteria into the upper urinary tract,
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Molecular Immunology 108 (2019) 56–67
and this shows the important role of flagella in the movement of the
bacteria from the bladder to the kidneys (Lane et al., 2007; Schwan,
2008; Wright et al., 2005). In addition, the important role of flagella has
been shown in different stages of biofilm formation in the urinary tract
(Pratt and Kolter, 1998).
2.2.6. LPS and capsule
The surface of UPEC strains composed of various polysaccharides.
The external side of outer membrane of UPEC contains lipopoly-
saccharide (LPS) that recognized as O antigen and usually is covered by
a capsular layer (K antigen) (Russo et al., 2009; Whitfield, 2006). There
is a high antigenic heterogeneity among O and K antigens of UPEC
strains (Whitfield and Roberts, 1999). For example, a high frequency of
the antigens O1, O2, O4, O6, O7, O8, O16, O18, O25, and O75 has been
observed among the UPEC isolates (Bidet et al., 2007; Lloyd et al.,
2007). The studies demonstrated that LPS and capsular polysaccharides
could be important virulence factors for evading of UPEC strains from
the host immune response mechanisms such as opsonophagocytosis,
complement-mediated bactericidal and killing by antimicrobial pep-
tides (Buckles et al., 2009; Whitfield, 2006). Some capsular types such
as K1 and K5 by showing a molecular mimicry to tissue components
neutralize the function of humoral response against urinary pathogens
(Johnson, 1991). In addition, LPS seems to have a role in colonization
of UPEC in the bladder, formation of IBC reservoirs, and resistance to
hydrophobic antibiotics (Aguiniga et al., 2016; Zhang et al., 2013).
3. Mechanisms of host defense against Urinary tract infections
In order to defense against urinary tract pathogens, including UPEC,
host utilizes different systems that are as follows:
3.1. Innate immune system
Innate immune system based on the presence of defense obstacles,
different compounds and antimicrobial peptides in the urine, such as
lysozyme, lactoferrin, defensins, lipocalin, cathelicidin, ribonuclease-7
and Tamm-Horsfall protein (neutralizing type 1 pili) is capable to
eliminate or exclude bacteria by different methods (Nielubowicz and
Mobley, 2010; Sivick and Mobley, 2010; Sivick et al., 2010). Further-
more, innate immune system based on the presence of defense cells
such as neutrophils, macrophages and Natural killer (NK) cells, pro-
duction of inflammatory cytokines in evoking the innate immune cells,
the presence of different components of the host complement system,
and the presence of scavenger receptors can prevent or control infection
by different mechanisms during the UTI process (Schilling et al., 2003;
Sivick et al., 2010; Weichhart et al., 2008).
3.2. Interaction between Toll like receptors and uropathogens
Interaction of Toll like receptors (TLRs) on the surface of the urinary
tract or immune cells with Pathogen-Associated Molecular Patterns
(PAMPs) in the uropathogens by production of cytokines, especially
inflammatory cytokines leads to develop the innate immune responses
and also directs these responses towards acquired immune system
(Nielubowicz and Mobley, 2010; Ragnarsdottir et al., 2008;
Vandewalle, 2008). It seems that TLRs such as TLR2, TLR4, TLR5 and
TLR11 have been implicated in defense against UTI pathogens
(Vandewalle, 2008). Expression of TLR2 as an immune receptor for
bacterial lipoteichoic acid or lipoprotein has been observed in different
parts of the kidneys such as proximal and distal-collecting tubules
(Takeda et al., 2003). TLR2 expression in the kidney cells by recogni-
tion of leptospiral outer membrane proteins resulted in the activation of
NF-κB and mitogen-activated protein kinases (MAPKs) (Yang et al.,
2006). One study has shown that TLR2-deficient mice exhibited a lower
level of inflammatory response and leukocyte infiltration as compared
to the control mice (Leemans et al., 2005). TLR4 is among the most
M.R. Asadi Karam, et al.
important receptors in inducing innate immunity against UTIs. After
entering the UPEC strains into the bladder, interaction of lipopoly-
saccharide (LPS) on the surface of these bacteria with TLR4 on the
bladder cells activates the inflammatory responses and evokes neu-
trophils to remove the bacteria from the urinary tract (Song and
Abraham, 2008). Furthermore, TLR4 signaling can be induced by an
LPS-independent mechanism. In this signaling pathway, binding of
adhesins of P and type 1 pili in UPEC strains to TLR4 on the urinary
tract resulted in the IL-6 and IL-8 cytokine response (Fischer et al.,
2006; Nielubowicz and Mobley, 2010). We previously demonstrated the
interaction between FimH adhesin of UPEC with TLR4 on bladder and
kidney cell lines, as well as induction of innate immunity by FimH-TLR4
interaction in animal model (Habibi et al., 2016). TLR5 as the receptor
of flagellin, the major structural protein of flagella in motile bacteria
has the ability to induce the innate immunity in bladder and kidney
against UTIs caused by UPEC (Andersen-Nissen et al., 2007; Rhee et al.,
2004). In this regard, TLR5-deficient mice showed a higher load of
bacteria in their bladders and kidneys in comparison to the wild type
mice that demonstrated the possible role of TLR5 in defense in the
urinary tract (Andersen-Nissen et al., 2007). TLR11 (recognizes the
Profilin-like protein in Toxoplasma gondii) is expressed in bladder and
kidney cells of mice, whereas it was not detected in humans
(Yarovinsky et al., 2005). Because of mice lacking TLR11 showed more
susceptibility to UTI pathogens (Zhang et al., 2004), TLR11 may have
important role in defense against uropathogens such as UPEC. Fur-
thermore, the lack of TLR11 in human may be one of the reasons for
high susceptibility of humans to UTIs (Vandewalle, 2008).
3.3. Acquired (adaptive) immunity
In the past, the role of acquired immunity due to T and B lympho-
cytes in protection against UTIs had been discussed and few studies had
been considered the role of specific immune response in defense against
UTIs (Nielubowicz and Mobley, 2010; Weichhart et al., 2008). In pre-
vious experiments, mice lacking T and B lymphocytes showed higher
sensitivity to UTIs compared to normal mice (Sivick and Mobley, 2010;
Weichhart et al., 2008). Transfer of T lymphocytes in spleen or inactive
transfer of serum from infected mice to naive mice resulted in protec-
tion against colonization by UPEC (Song and Abraham, 2008;
Thumbikat et al., 2006). In another study, the γδ T-lymphocyte-defi-
cient mice showed higher sensitivity to UTIs (Jones-Carson et al.,
1999). However, the recent studies, including vaccine studies per-
formed by us have shown humoral and cell mediated immunity by
producing different cytokines such as interferon gamma (IFN-γ), in-
terleukin-4 and interleukin-17 have important roles in eradicating UTIs
(Asadi Karam et al., 2013; Habibi et al., 2015a, b; Karam et al., 2013,
2016). On the other hand, it seems that the host defense responses is
different in the kidney and bladder, so that mucosal defense including
the humoral responses induced by secretoryIgA is maybe the most ef-
fective defense for inhibiting colonization of the bacteria in the bladder,
but systemic defense produced by T lymphocytes is the most important
immune system in the kidneys (Sivick and Mobley, 2010). Furthermore,
resolution of UTI and its non-recurrence in 75% of UTI patients can be
due to the production of acquired immunity against UPEC after an in-
itial infection. However, some also believe that the defect in producing
the secretory IgA antibodies is one of the main causes of the recurrence
in patients causing to UTIs (Song and Abraham, 2008; Weichhart et al.,
2008).
4. Antibiotic therapy of UTI patients and emergence of resistance
4.1. Antibiotic therapy of different types of UTI
The routine therapy of UTIs is performed based on the use of anti-
biotics, and appropriate antibiotics should be selected by considering
the characteristics such as the antibiotic susceptibility pattern of the
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Molecular Immunology 108 (2019) 56–67
causative isolate, infection type (community-acquired or hospital-ac-
quired infection), the patient's conditions including age, gender, history
of allergy, underlying diseases, previous antibiotic consumption, taking
the other drugs, history of previous UTIs, site of UTI (bladder, kidney or
prostate) and also the pathogenic or normal flora of the causative iso-
late (De Francesco et al., 2007; Ejrnaes, 2011; Walker et al., 2016).
Trimethoprim, β-lactams and nitrofurantoin antibiotics have been se-
lective drugs for treatment of uncomplicated UTIs in many countries
(Ejrnaes, 2011; Vachhani et al., 2015). Of course, the use of fluor-
oquinolones is among the selective drugs for treatment of complicated
and uncomplicated UTIs in some countries (Fasugba et al., 2015). The
Infectious Diseases Society of America has recommended fosfomycin
and nitrofurantoin drugs for treatment of patients with uncomplicated
UTIs, even in causing to UTI with resistant strains. In some complicated
UTIs, antibiotic treatment is difficult, which treatment these type of
infections is done according to the conditions of disease and the pa-
tients (Walker et al., 2016). Antibiotic therapy for recurrent UTIs has
always been discussed, and if not correctly selected, antibiotic not only
don't destroy the reservoirs of bacteria, it can also act as a shelter for the
survival of the bacteria in the bladder cells (Terlizzi et al., 2017). De-
spite the lack of comprehensive studies, it is recommended that some
studies will perform to determine the effectiveness of antibiotics and
their behaviors in eliminating IBC forms of UPEC as the major cause of
recurrent UTIs, which would certainly be helpful in treatment of UTI
patients in the future.
4.2. Dissemination of antibiotic resistance among the UPEC strains
Routinely, different studies are performed on the evaluation of an-
tibiotic resistance in UPEC isolated from UTI patient’s in different
countries and their results have been published in different journals. By
reviewing these published articles, it can be concluded that according
to the time of study and the geographic area, different rates of re-
sistance to antibiotics have been reported. However, these studies,
especially in developing countries show the increasing resistance to the
antibiotics that some of them have been reported as multi-drug re-
sistance (MDR) (Sanchez et al., 2016). There are different risk factors
associated with acquiring multi-drug resistance including the use of
antibiotics before causing to UTI, hospitalization for a long time, the
use of medical devices such as urinary catheters, having underlying
diseases as well as an old age (Ejrnaes, 2011; Walker et al., 2016). In a
systematic review study performed by Hadifar et al. (Hadifar et al.,
2017) in Iran in 2017, the average prevalence of UPEC strains with
multiple resistance (resistance to three or more antibiotic classes) was
reported about 50% in different regions of Iran. In India, it seems that in
2007–2008, the prevalence of UPEC with multiple resistances was less
than 50%, but since 2011, the multiple resistance has increased up to
80% (Dash et al., 2012; Hadifar et al., 2017; Hasan et al., 2007). In
Pakistan, since 2011, the prevalence of multi-drug resistant strains has
been reported more than 65% (Ali et al., 2014; Sabir et al., 2014).
Reports have shown that even in developed countries such as the
United States, infection with multi-resistant UPEC strains among the
outpatients have increased from 9% in 2001 to about 17% in 2010
(Sanchez et al., 2014). These reports show that the resistance of UPEC
strains to the routine therapeuticantibiotics of UTIs such as beta-lac-
tams, nalidixic acid, co-trimoxazole, ciprofloxacin and tetracycline has
increased, which could be due to the excessive use of antibiotics, self-
medication, and consumption of animal products that antibiotics have
been added to them (Caracciolo et al., 2011; Farshad et al., 2012;
Mukherjee et al., 2013; Walker et al., 2016). Resistance to these anti-
biotics in some countries resulted in the consumption of alternative
antibiotics such as fluoroquinolones and extended-spectrum cephalos-
porins, which a significant and alarming resistance to them also is re-
ported from different countries (Molina-López et al., 2011; Neamati
et al., 2015; Pignanelli et al., 2013). In Europe, resistance to fluor-
oquinolones and cephalosporins is reported 22% and 12%, respectively.
M.R. Asadi Karam, et al.
In the United States, the prevalence of fluoroquinolone-resistant UPEC
strains was reported about 31% among the hospitalized patients
(Edelsberg et al., 2014). Of course, one of the reasons that resulted in
the variable reports of antibiotic resistance among the UTI pathogens is
the lack of same methods in performing the antimicrobial susceptibility
tests in the countries. By equalizing these methods according to the
Clinical and Laboratory Standards Institute (CLSI) recommendations, it
can be expected that more reliable results will be obtained from reports
of antibiotic resistance in countries that can be used to select the best
therapeutic antibiotics against UTIs.
4.3. Mechanisms of resistance acquisition to antibiotics in UPEC strains
Antibiotic resistance in UPEC strains can be acquired through dif-
ferent mechanisms, which often occur due to the acquisition of re-
sistance genes via mobilegenetic elements such as plasmids, transpo-
sons, gene cassettes in the integrons, or changes in regulatory locus,
called mar (multiple antibiotic resistance locus) on the chromosome of
the bacteria (Paniagua-Contreras et al., 2017; Tong et al., 2017).
4.3.1. Acquisition of resistance to β-lactam antibiotics in UPEC
Resistance to β-lactam antibiotics, such as ampicillin, amoxicillin
and cephalosporins usually is acquired due to the production of various
types of β-lactamase enzymes (Dashti et al., 2006). A group of β-lac-
tamases, called Extended Spectrum β-Lactamases (ESBLs) is produced
after mutations in the ancestral enzymes blaTEM-1, blaTEM-2, and
blaSHV-1, and are often transferred by plasmids (Dashti et al., 2006;
Hawkey and Munday, 2004). Since the early 2000s, a new β-lactamase,
called blaCTX-M has also been identified in UPEC strains (Barguigua
et al., 2011). In our recent study, three classes of β-lactamase enzymes
including TEM, SHV and CTX-M were observed among the UPEC iso-
lated from different hospitals in Iran (Shahbazi et al., 2018). Further-
more, in accordance with our results, the most common ESBLs reported
from Western and Asian countries have been TEM and SHV β-lacta-
mases (Paterson and Bonomo, 2005; Shahbazi et al., 2018). ESBL en-
zymes are able to encode resistance to all β-lactam antibiotics expect for
carbapenems, cephamycins and β-lactamase inhibitors (Baudry et al.,
2009). In accordance with our recent studies, it was found that UPEC
isolates with the ability of producing ESBL enzymes showed more rates
of resistance to β-lactams, aminoglycosides, and quinolone families of
antibiotics as compared to the non-producing ESBL strains (Barguigua
et al., 2011; Shahbazi et al., 2018). Therefore, it could be concluded
that nowadays, increasing dissemination of ESBLs among the UPEC
population should be considered as an important challenge in treatment
of UTIs caused by UPEC strains.
4.3.2. Acquisition of resistance to carbapenems in UPEC
Carbapenems have been among the most effective antibiotics
against ESBL-producing isolates. Resistance to carbapenems is pro-
duced due to several mechanisms including decreased permeability,
overexpression of efflux pumps, and secretion of carbapenem-hydro-
lyzing enzymes called carbapenemases (Fusté et al., 2013; Patel and
Bonomo, 2013). Different types of carbapenemases such as VIM, IMP,
NDM, SPM, GIM, SIM, AIM, DIM, OXA-48 and KPC have been identi-
fied. Among them, most of the NDM-1 producing bacteria showed high
resistance to different classes of antibiotics, including beta-lactams and
carbapenems and these enzymes are often seen in developing countries
such as India and Pakistan (Patel and Bonomo, 2013). Of course, in our
recent studies, no carbapenemase gene has been observed in the UPEC
isolates, suggesting the low rate of these enzymes among the UPEC
isolates in Iran (Shahbazi et al., 2018).
4.3.3. Acquisition of resistance to quinolones in UPEC
Quinolones and fluoroquinolones are among the most common
antibiotics in treatment of UTIs and their excessive use has led to an
increased resistance in UTI pathogens (Zurfluh et al., 2014). Mutations
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Molecular Immunology 108 (2019) 56–67
in DNA topoisomerases II and IV, decreased uptake of the antibiotics,
extrusion of antibiotics via efflux pumps, and plasmid-mediated qui-
nolone resistance (PMQR) genes are the major resistance mechanisms
to these antibiotics (Strahilevitz et al., 2009; Zurfluh et al., 2014). Qnr
genes (qnrA, qnrB and qnrC) are the most important PMQR genes that
cause the antibiotic resistance by inhibiting the binding of quinolones
to DNA gyrase and topoisomerases (Robicsek et al., 2006). Qnr genes
due to locating on different integrons resulted in the disseminated re-
sistance in UPEC strains (Robicsek et al., 2006). In studies conducted by
our research team, a variety of qnr genes, especially qnrB and qnrC
genes have been observed in UPEC isolated from Iranian UTI patients
(Shahbazi et al., 2018).
In summary about studies on antibiotic resistance, it can be sug-
gested that monitoring the microbial resistance should be done peri-
odically and continuously in different countries in order to perform the
best antibiotic therapy against these infections and prevent or decrease
the antibiotic resistance among the causative strains.
5. Vaccine candidates designed against urinary tract infections
Reduced effectiveness of antibiotics due to the increased antibiotic
resistance and their side effects on normal flora of the human body led
to continue the researches to find the new therapeutic strategies against
UTIs, which the most important of them is development of effective
vaccines (Gupta et al., 2001; Moura et al., 2009; O’Brien et al., 2016).
Development an effective vaccine against UPEC strains will have an
important role in reducing the mortality and morbidity rate of patients
as well as reducing economic costs. Production of an effective UPEC
vaccine is challenging for several reasons: 1) Heterogeneity of UPEC
strains. 2) The probability of the side effects on the normal flora of the
intestine. 3) Dependence of UTIs to the expression of multiple virulence
factors of UPEC strains (O’Brien et al., 2016). Since UPEC strains utilize
a variety of virulence factors, therefore, it seems that an effective
vaccine should be able to provide a protective immune response against
virulence factors that are expressed in different stages of UTI develop-
ment, such as colonization, invasion, and the formation of IBC re-
servoirs (Brumbaugh and Mobley, 2012; McLellan and Hunstad, 2016).
Vaccines that so far have been developed against UTIs can be
classified into two groups: 1) Cell-based vaccines (killed or live-atte-
nuated vaccines) and 2) Antigen-based vaccines including subunits,
toxin-based and conjugate vaccines (Brumbaugh and Mobley, 2012).
Each of these vaccine groups has some advantages and disadvantages.
One of the disadvantages of purified antigens compared to whole-cell
based vaccines is the absence of strong and long-term immune re-
sponses that by using the components named adjuvants it is possible to
increase their immunogenicity (Choubini et al., 2018; Skwarczynski
and Toth, 2016).
5.1. Development of cell-based vaccines
The whole cell or cell-lysate based vaccines can be considered as the
only vaccines available on the market. Solco-Urovac, as the first killed
polymicrobial vaccine, consisted of 10 strains of killed uropathogens (6
UPEC strains, 1 strain of each Proteus mirabilis, Klebsiella pneumoniae,
Morganella morganii and Enterococcus faecalis) protected mice for
higher than 20 weeks against the challenge with different strains (Kruze
et al., 1992, 1989), and also could reduce the occurrence of recurrent
UTIs in women (Grischke and Rüttgers, 1987). This vaccine by induc-
tion of strong mucosal responses from the vaginal route protected
women against recurrent UTI. Furthermore, the vaginal inoculation of
this vaccine in 6 doses could significantly reduce the number of UTIs
caused by UPEC strains (Uehling et al., 1997, 2001; Uehling et al.,
2003). Of course, Urovac in the volunteers had side effects such as
redness at the injection site, pressure, pain, fever, burning, bleeding,
vaginal itching, and nausea, which limited the use of the vaccine as a
general vaccine (Grischke and Rüttgers, 1987; Hopkins et al., 2007).
M.R. Asadi Karam, et al.
Uro-Vaxom (OM89) has been marketed for the first time in Switzerland
as a vaccine composed of membrane proteins of 18 strains of UPEC, and
has been used in other 40 countries. The formulation of this vaccine is
in the oral capsule form, and studies have shown that the vaccine has
been able to effectively prevent recurrent UTIs in women (Bauer et al.,
2005, 2002; Schulman et al., 1993). The vaccine has shown few side
effects in patients, but its main disadvantage is continuous use of the
drug for three months, which makes its use difficult for all patients (Kim
et al., 2010). Urvakol and Urostim as killed vaccines are a mixture of
killed uropathogens, such as UPEC. Formulation of these vaccines is in
the form of daily oral tablets that showed high stimulation of the sys-
temic and mucosal responses, as well as the production of cytokines
(Koukalova et al., 1999a, b; Marinova et al., 2005). Since the clinical
trial phase studies on these vaccines have not yet been completed and
no results have been reported, the ability of these vaccines to protect
against recurrence of UTI has not been proven.
Among the first live-attenuated vaccines, a vaccine named as CP923
was developed using the mutations in capsule and O antigen from LPS
in UPEC strain. Intranasal inoculation of killed CP923 strain with for-
malin produced significant humoral immune responses in serum, but
could not protect the mice in a sepsis infectious model (Russo et al.,
2007, 2009). The reason for this lack of protection was the presence of
surface polysaccharides in the CP923 strain and other UPEC strains
which covered the lower epitopes and reduced the effectiveness of
antibody responses produced against non-polysaccharide epitopes
(Russo et al., 2007, 2009). Among the other live-attenuated vaccines, a
vaccine called NU14 ΔwaaL developed using a deletion in the gene
encoding O antigen ligase, which inhibited the bacterial colonization in
the urinary tract. The inoculation of this vaccine into the bladder of
mice protected the bladder and inhibited the stability of infections
caused by different strains of UPEC. Disadvantages of this vaccine were
its instability after inoculation in the bladder of mice as well as lack of
kidney protection (Billips et al., 2009).
5.2. Development of antigen-based vaccines
5.2.1. Capsular or LPS-based vaccines
The capsule-forming surface polysaccharides (K antigen) and lipo-
polysaccharides (O antigen) were among the first vaccines developed
against UPEC strains. These vaccines could provide protective immune
responses in mice model, but despite the high diversity of these anti-
gens, designing a vaccine that can cover all UPEC serotypes is chal-
lenging. The other disadvantage of capsular polysaccharides is their low
immunogenicity in induction of acquired immunity (Johnson, 1991;
O’Brien et al., 2016). Despite this, a conjugate vaccine composed of O
antigen of UPEC serotypes resulted in the reduction of UTI in women
with recurrent UTI (Huttner et al., 2017).
5.2.2. Fimbrial and afimbrial adhesin-based vaccines
Since Pili play an important role in the binding and colonization of
UPEC strains to the urinary tract, and also are presented in the surface
of bacteria to be available for the immune system of host, various
studies have been carried out on adhesin-based vaccines (Brumbaugh
and Mobley, 2012; Spurbeck et al., 2011). FimH adhesin from type 1
pili has devoted most attention to itself, by having the characteristics of
an ideal vaccine candidate including high immunogenicity, conserva-
tion of the antigenic structure among different strains of UPEC, the high
prevalence among different strains of UPEC, high expression at the site
of infection, and the important role in the pathogenesis (Asadi Karam
et al., 2013; Langermann et al., 2001; Thankavel et al., 1997). A
truncated FimH vaccine and a complex of full length of FimH protein
with chaperone FimC (FimCH vaccine) were able to reduce the colo-
nization of different UPEC strains in the bladder of mice. In the next
stage, the injection of FimCH vaccine with Freund's adjuvant led to
increase in the IgG response in the serum and protected mice nearly
100% against bacterial colonization in the bladder (Langermann et al.,
61
Molecular Immunology 108 (2019) 56–67
1997). Evaluation the FimH vaccine with Alum and MF59 as human
adjuvants had also successful results. Evaluating the function of FimCH
admixed with MF59 adjuvant on the primate models produced sig-
nificant IgG levels in serum and vagina and protected the monkeys
against infection (Langermann et al., 2001, 2000). The vaccine then
was entered to phase II clinical trial, but several years later, it was
announced that the project was stopped due to the ineffectiveness of the
vaccine (Brumbaugh and Mobley, 2012; O’Brien et al., 2016). There are
several reasons for the failure of FimH vaccine: 1) The promoter of type
1 pili is located on an invertible element that switches expression of
FimH between on and off, thus these variations in the expression of type
1 pili may cause the UPEC strain not to be recognized by the acquired
immune system (Eisenstein, 1981; Nowicki et al., 1984). 2) The anti-
bodies developed against FimH do not target its mannose-binding re-
gion or by changing the tertiary structure of FimH increases its binding
to mannosylated receptors on the surface of epithelial cells
(Tchesnokova et al., 2011). 3) Differences in the expression pattern of
the virulence genes in animal and human models may be the other
reason for this failure. This suggests that the design of an effective UTI
vaccine for human use requires further studies to evaluate the expres-
sion of the virulence genes of uropathogens in human.
As mentioned, interaction of TLRs especially TLR2, TLR4, TLR5 and
TLR11 on the surface of immune cells and urinary epithelial cells with
PAMPs on uropathogens results in the induction of innate and adaptive
immunity (Vandewalle, 2008). Therefore, stimulation of these TLRs can
be performed by their specific ligands as innate adjuvants to induce
strong immune responses in the subunit vaccines (Hagan and Mobley,
2007). In several recent studies, our research team has used these TLR
ligands to induce the immune system in designed UTI vaccines (Asadi
Karam et al., 2013; Habibi et al., 2015a, b; Karam et al., 2013, 2016). In
one of these studies, fusion of FimH adhesin to flagellin (FliC) of UPEC
strain as a TLR5 ligand was able to induce significant humoral and
cellular responses and protected mice against UTI in a bladder chal-
lenge (Asadi Karam et al., 2013). Of course, using the Montanide as a
commercial adjuvant with formulation water in oil could also induce
significant immune responses against fusion FimH.FliC and protected
mice in an experimental challenge (Asadi Karam et al., 2013). Another
major finding of our previous studies was that the IgG humoral re-
sponse was maintained at a high level for about 8 months after the first
vaccine dose, which could be useful for the eradication of intracellular
reservoirs (IBCs) of UPEC strains in recurrent UTIs, and cover one of the
disadvantages of the previous vaccines that didn’t induce long-term
immune responses (Asadi Karam et al., 2013; Habibi et al., 2015a, b;
Karam et al., 2013, 2016). Of course, more studies are currently un-
derway on the vaccine candidates to evaluate them in the monkey
model.
Despite the role of cellular immunity in protection against UTIs,
especially eradication of IBC reservoirs of UPEC, and neglecting its role
in some studies (Wieser et al., 2010), we evaluated the efficacy of fusion
FimH.FliC admixed with Cholera toxin (CT) adjuvant to stimulate cel-
lular immune responses (Karam et al., 2016). This vaccine combination
from the intranasal route could induce humoral and a mixed of Th1 and
Th2 responses with a tendency towards Th1 (cellular immunity) that
resulted in a significant protection against experimental infection. One
of interesting results of this study was that despite the lower amount of
protein and antigen used in the intranasal route, cellular responses were
stronger than the subcutaneous route (Karam et al., 2016). Further-
more, a DNA vaccine composed of adhesin components of UPEC strains
was used to induce cellular immune responses against UTI. In defini-
tion, DNA vaccine is composed of a prokaryotic plasmid vector that
encodes the inserted antigen under the control of a eukaryotic promoter
(Saade and Petrovsky, 2012). DNA vaccine has the advantage of in-
duction both humoral and cellular immune responses (Cui, 2005). In
this regard, Fooladi et al. (Fooladi et al., 2014) showed that FimH as a
DNA vaccine significantly increased cell proliferation with production
of IFN-γ cytokine and reduced the bacterial load in a bladder challenge
M.R. Asadi Karam, et al.
model.
Since UTI, especially in catheterized patients can be occurred in a
polymicrobial form, thus, design of a polyvalent vaccine can be an ef-
fective way to deal with these polymicrobial infections (Habibi et al.,
2015a, b). In most cases, Escherichia coli and Proteus mirabilis are
common uropathogens in these polymicrobial communications. In the
recent studies done by our research team, a fusion of FimH and MrpH
adhesins of UPEC and Proteus mirabilis in combination with Monopho-
sphoryl Lipid A (MPL) adjuvant as a TLR4 ligand could induce sig-
nificant humoral and cellular responses and significantly reduced the
load of these bacteria in the bladder and kidneys (Habibi et al., 2015a,
b). Further studies on this vaccine candidate are ongoing.
A PapG pili-based subunit vaccine could inhibit UPEC in the kidney
of mice and monkey models (Roberts et al., 2004). The disadvantage of
this vaccine was the lack of protection in the bladder, which is probably
due to the absence of other pili involved in the bacterial colonization in
the bladder, such as type I pili (Roberts et al., 2004). It seems that the
problem can be solved by combining the PapG with other antigens in a
polyvalent form. On the other hand, despite there are different ser-
otypes of P fimbriae among the UPEC strains, it is better to use a
mixture of common serotypes of P fimbriae.
In assessment another vaccine based on Dr fimbriae, its purified
protein with Freund's adjuvant resulted in high production of antibody
responses and a reduction in mortality of mice (Goluszko et al., 2005).
Although, the vaccine reduced colonization of bacteria in the bladder
and kidneys of mice compared to the control mice, but this reduction
was not significant (Goluszko et al., 2005). S fimbriae have also been
studied as a vaccine candidate, but it has not been able to protect mice
against lethal sepsis in a mice model (Stins et al., 1994).
Among the afimbrial adhesins as vaccine candidate, mutation in the
gene encoding TosA in UPEC strain has caused a defect in the ability of
UPEC colonization in the urinary tract. But, vaccination of mice with
this protein could not protect the mice against UTI (Vigil et al., 2011,
2012). In another study, the vaccination of mice with the FdeC adhesin
from the intranasal route only protected the colonization of UPEC strain
in the kidneys and had no effect on the protection of the mice bladder
(Nesta et al., 2012). In another study, Iha iron-regulating adhesin could
significantly protect the UPEC strain in a mice model (Johnson et al.,
2005).
Auto-transporters such as UpaG from UPEC strains as adhesin factor
have also been studied as vaccine candidates. In a sepsis model, im-
munization of mice with the UpaG protein also provided protection
after active and passive immunization (Durant et al., 2007; Valle et al.,
2008). The efficacy of a variant of Ag43, Ag43a, also is shown in long-
term persistence of UPEC CFT073 strain in the bladder of challenged
mice (Ulett et al., 2007). Furthermore, we are going to evaluate the
immunogenicity and efficacy of Ag43a in protection against experi-
mental UTI in an animal model and their results will be published in the
future.
5.2.3. Iron scavenger receptors-based vaccines
UPEC strains utilize multiple iron absorption systems and these
factors have the characteristics of a vaccine candidate, including ex-
pression on the surface of bacteria to be available for immune re-
sponses, the high prevalence among UPEC strains, and expression
during infection (Garcia et al., 2011; Habibi et al., 2017). So far, a
number of iron absorption receptors of UPEC strains have been in-
vestigated as UTI vaccine candidates (Alteri and Mobley, 2007). Studies
have shown that mutation in heme acquisition receptor ChuA and
aerobactin IutA in the UPEC strains reduced the number of bacteria in
the bladder of mice (Torres et al., 2001). In another study, among the
iron scavenger receptors tested, IutA, siderophore receptor IreA and
heme acquisition receptor Hma protected the mice against UTI. In this
study, Hma and IreA showed the highest protection in the kidney and
bladder, respectively, while IutA protected both the bladder and kid-
neys (Alteri et al., 2009). Russo et al. (Russo et al., 2003) showed that
62
Molecular Immunology 108 (2019) 56–67
immunization of mice with denatured form IroN, despite the strong
humoral responses only protected the kidneys against UTI. In another
study, IroN injected with Freund's adjuvant showed about 82% and
79% protection after active and passive immunization, respectively,
whereas this rate of protection for yersiniabactin FyuA admixed with
Freund's was 53% and 72% post active and passive immunization, re-
spectively (Durant et al., 2007). In another study done by Alteri et al.
(Alteri et al., 2009), the intranasal immunization with IutA conjugated
with Cholera toxin by production of significant IgA protected mice
against UTI in the bladder and kidneys. In another study done by our
research team, injection of FyuA protein with Alum adjuvant produced
strong systemic humoral responses from subcutaneous route, which
resulted in protection against UPEC infection (Habibi et al., 2017). Mike
et al. (Mike et al., 2016) indicated that conjugation of yersiniabactin
and aerobactin with cationized BSA was able to produce acquired im-
mune responses and protected mice against UTI.
In some studies, surface components of iron absorption receptors
have been investigated in the multi-epitope vaccine forms. In one of
these studies, external loops of two IroN and IutA proteins were syn-
thetized as a linear peptide and the results showed that immunization of
mice with this peptide reduced colonization of UPEC in the kidneys
(Alteri et al., 2009). In other study, two multi-epitope subunit vaccines
were made from surface domains of iron adsorption receptors IutA, Iha,
FyuA, IroN, IreA and ChuA and in conjugation with Cholera toxin
protected the mice against peritonitis. Furthermore, the first structure
(Vol 1) of this designed vaccine including the domains of FyuA, IutA,
Iha and another protein from UPEC was able to reduce bacterial colo-
nization in the spleen of mice (Wieser et al., 2010).
Since one of the disadvantages of subunit recombinant vaccines is
low immunogenicity, their application with delivery systems can in-
duce immune responses and increase the longevity of these responses.
The use of these delivery systems, such as Salmonella, for UTI vaccines
is ongoing by our research team, which their results will be published in
the future. In another study, the efficacy of a multi-epitope vaccine
composed of iron absorption factors had been evaluated using a vaccine
delivery system in Salmonella, which IroN, IutA, IreA and FyuA showed
significant protection in the urinary tract of mice (Wieser et al., 2012).
In summary about the use of iron scavenger receptors as UTI vac-
cine, it can be concluded that due to the diversity of these factors in
UPEC strains, it is better to use a mixture of these factors with other
virulence characteristics of UPEC strains such as colonization compo-
nents to reach a more effective vaccine against UTIs (Garcia et al.,
2011; Hagan et al., 2010).
5.2.4. Toxins-based vaccines
The first vaccine based on UPEC toxins was hemolysin (HlyA) that
was purified from the supernatant of cultured UPEC and resulted in a
decrease in kidney damage of mice, but this reduction was not sig-
nificant (O’Hanley et al., 1991). In another study, recombinant hemo-
lysin (ecp_3827 candidate) amplified from UPEC strain 536 provided
76% protection in a sepsis mice model (Moriel et al., 2010). In several
researches, the mutations in cnf1 and hlyA toxin genes, or both of them
in the UPEC strain reduced cystitis in mice compared to the control
group and mutation in the cnf1 gene reduced the severity of prostatitis
(Rippere-Lampe et al., 2001a, b; Smith et al., 2008). In another study in
which mice groups were vaccinated subcutaneously with toxoids of
hlyA and CNF1, mice vaccinated with hlyA induced a significant anti-
body in serum and after the challenge of mice, the load of bacteria was
significantly decreased in the urine and bladder. In the mice group
vaccinated with CNF1, although the antibody titer was increased in
serum, no reduction in the bacterial number was found in the urine and
kidneys (Smith et al., 2015). One of the reasons for the inefficiency of
vaccination with CNF1 could be the inadequate titer of neutralizing
antibodies in the bladder (Smith et al., 2015).
Auto-transporter toxins such as Vat from UPEC strains have also
been studied as vaccine targets. In a study, in the sepsis model, Vat
M.R. Asadi Karam, et al.
resulted in 32% and 78% protection after active and passive im-
munization, respectively (Durant et al., 2007).
Overall, it can be concluded that optimization of vaccines based on
the UPEC toxins could be helpful in development an effective vaccine
against UTI. Designing the new vaccine candidates based on the dif-
ferent epitopes of these toxins is underway by our research team.
6. Using the other therapeutic strategies against UTIs
6.1. Probiotics as a therapeutic strategy against UTI
The use of probiotics, especially strains of Lactobacilli, has today
been proposed as a therapeutic strategy, especially in patients with
recurrent UTIs, that in some cases, hopeful results have been observed
(Shim et al., 2016; Zacche and Giarenis, 2016). Probiotics may com-
pensate the alterations in the bacterial normal flora (microbiota) of
vagina in women with recurrent UTI, produce factors such as anti-ad-
hesive molecules and stimulate the immune responses in the vagina to
inhibit or decrease the bacterial colonization in the UTI patients (Czaja
et al., 2009; O’Brien et al., 2016). Of course, further studies are needed
to demonstrate the effects of probiotics as a therapeutic strategy against
UTIs.
6.2. Estrogens as a therapeutic strategy against UTI
The use of the ability of estrogens, especially in maintaining the
structure of bladder epithelial cells and their effects on the formation of
IBC reservoirs of UPEC is one of other therapeutic strategies against
UTIs (Terlizzi et al., 2017). Estrogens can defense against UTI patho-
gens by production of antimicrobial peptides and reduction the epi-
thelial exfoliation of the urinary tract (Luthje et al., 2013). In one of
these studies, estrogens have prevented from the recurrent infection,
especially in combination with other compounds such as hyalur-
onicacid and chondroitin sulfate (Torella et al., 2016).
6.3. Pilicides and Curlicides as a therapeutic strategy against UTI
Pilicide compounds have been used in studies as inhibitors of bac-
terial pili to bind to target cells. These compounds are derivative of
amino acids or pyridine compounds that have been able to decrease
haemagglutination in in vitro by neutralization the type 1 and P pili and
also to inhibit the formation of bacterial biofilms (Pinkner et al., 2006;
Svensson et al., 2001). Curlicide compounds also showed the ability to
block the assembly of type 1 pili and formation of biofilm (Cegelski
et al., 2009). In one study, the ability of a Curlicide compound named as
FN075 was shown in reduction the biofilm formation and virulence of
UPEC strain in a bladder challenge model in mice (Cegelski et al.,
2009).
6.4. Antimicrobial peptides as therapeutic strategies against UTI
Nowadays, antimicrobial peptides are also proposed as the alter-
natives of antibiotics. These compounds have hydrophobic short chains
of amino acids with a positive charge in their structures that can target
different components of the bacterial cells (Lo and Lange, 2015). At
first, the effectiveness of lactoferrin and its derived peptides was shown
in reduction of bacterial load in the bladder of mice (Haversen et al.,
2000). The effectiveness of Tachyplesin III was also showed as an an-
timicrobial peptide in inhibiting biofilm formation (Minardi et al.,
2007). Up to now, different antimicrobial peptides are designed, which
have been reviewed in the cited references (Becknell et al., 2015; Lo
and Lange, 2015). Of course, cautions should be considered for using
these peptides in treatment of UTI patients, for example, high con-
centrations of antimicrobial peptide LL-37 resulted in the inflammation
in the urinary epithelial cells and cell death (Becknell et al., 2015).
Different in vivo and in vitro studies are ongoing by our research team to
63
Molecular Immunology 108 (2019) 56–67
investigate the effectiveness of several designed antimicrobial peptides
against UTIs caused by UPEC (data not published).
7. Conclusion
Nowadays, urinary tract infections, especially UTIs caused by UPEC
strains are one of the most common infections in the societies that the
complicated and recurrent UTI forms can complicate the process of
treatment. The extensive use of antibiotics in treatment of UTIs has
resulted in increasing resistance to them among UPEC strains, which
will result in more challenges for treatment of these infections and even
other bacterial infections in the future. Therefore, new therapeutic
strategies such as effective vaccines or antimicrobial peptides can be
used at least for the populations at high risk of the disease, such as those
with recurrent infections and those who are hospitalized for a long
time. Nowadays, despite the vaccine candidates developed against
UTIs, no effective UTI vaccine has been developed. Thus, more studies
on novel antigens, adjuvants, route of administration, and the use of
secretion systems are needed to reach an effective and general vaccine
against UTIs.
Acknowledgements
The study was supported by Pasteur Institute of Iran.
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