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Uropathogenic Escherichia coli (UPEC) deploy an array of virulence factors to successfully establish urinary tract infections. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Two-component signaling systems (TCSs) are a major mechanism by which bacteria sense environmental cues and respond by initiating adaptive responses. Here, we began this study by characterizing a novel TCS (C3564/C3565, herein renamed orhK/orhR for oxidative resistance and hemolysis kinase/regulator) that is encoded on a UPEC pathogenicity island, using bioinformatic and biochemical approaches. A prevalence analysis indicates that orhK/orhR is highly associated with the UPEC pathotype, and it rarely occurs in other E. coli pathotypes tested. We then demonstrated that OrhK/OrhR directly activates the expression of a putative methionine sulfoxide reductase system (C3566/C3567) and hemolysin (HlyA) in response to host-derived hydrogen peroxide (H2O2) exposure. OrhK/OrhR increases UPEC resistance to H2O2 in vitro and survival in macrophages in cell culture via C3566/C3567. Additionally, OrhK/OrhR mediates hemolysin-induced renal epithelial cell and macrophage death via a pyroptosis pathway. Reducing intracellular H2O2 production by a chemical inhibitor impaired OrhK/OrhR-mediated activation of c3566-c3567 and hlyA. We also uncovered that UPEC links the two key virulence traits by cotranscribing the c3566-c3567 and hlyCABD operons. Taken together, our data suggest a paradigm in which a signal transduction system coordinates both bacterial pathogen defensive and offensive traits in the presence of host-derived signals; and this exquisite mechanism likely contributes to hemolysin-induced severe pathological outcomes., Competing Interests: The authors have declared that no competing interests exist.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells., Competing Interests: The authors have declared that no competing interests exist.

The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen., Competing Interests: The authors have declared that no competing interests exist.

Background: Schistosoma haematobium infections are responsible for significant urinary tract (UT) complications. Schistosomiasis control programs aim to reduce morbidity, yet the extent of morbidity in preschool-aged children and the impact of treatment on morbidity reduction are not well studied., Methodology: Our study was embedded in a randomized, placebo-controlled, single-blind trial in Côte d'Ivoire, which evaluated the efficacy and safety of three doses (20, 40 and 60 mg/kg) of praziquantel in school-aged (SAC) and preschool-aged (PSAC) children infected with S. haematobium. Enrolled children were invited to participate in an ultrasound examination prior and six months after treatment. At these time points 3 urine samples were collected for parasitological and clinical examinations., Principal Findings: 162 PSAC and 141 SAC participated in the ultrasound examination at baseline, of which 128 PSAC and 122 SAC were present at follow-up. At baseline 43% (70/162) of PSAC had UT morbidity, mostly at bladder level and 7% had hydronephrosis. 67% (94/141) of SAC revealed mainly moderate UT pathology, 4% presented pseudopolyps on the bladder wall, and 6% had pyelectasis. At follow up, 45% of PSAC and 58% of SAC were S. haematobium positive, mostly harboring light infection intensities (41% and 51%, respectively). Microhematuria was present in 33% of PSAC and 42% of SAC and leukocyturia in 53% and 40% of PSAC and SAC, respectively. 50% (64/128) of PSAC and 58% (71/122) of SAC presented urinary tract morbidity, which was mainly mild. A significant correlation (p<0.05) was observed between praziquantel treatment and reversal of S. haematobium induced morbidity. Progression of UT pathology decreased with increasing praziquantel dosages. A worsening of morbidity was observed among children in the placebo group., Conclusion/significance: Bladder morbidity is widespread among PSAC. Praziquantel treatment is significantly associated with the reversal of S. haematobium induced morbidity, which underscores the importance of preventive chemotherapy programs. These programs should be expanded to PSAC to prevent or decrease the prevalence of morbidity in young children. This trial is registered as an International Standard Randomized Controlled Trial, number ISRCTN15280205.

Streptococcus agalactiae causes urinary tract infection (UTI) in pregnant adults, non-pregnant adults, immune-compromised individuals and the elderly. The pathogenesis of S. agalactiae UTI in distinct patient populations is poorly understood. In this study, we used murine models of UTI incorporating young mice, aged and dam mice to show that uropathogenic S. agalactiae causes bacteriuria at significantly higher levels in aged mice compared to young mice and this occurs coincident with equivalent levels of bladder tissue colonisation at 24 h post-infection (p.i.). In addition, aged mice exhibited significantly higher bacteriuria burdens at 48 h compared to young mice, confirming a divergent pattern of bacterial colonization in the urinary tract of aged and young mice. Multiparous mice, in contrast, exhibited significantly lower urinary titres of S. agalactiae compared to age-matched nulliparous mice suggesting that parity enhances the ability of the host to control S. agalactiae bacteriuria. Additionally, we show that both age and parity alter the expression levels of several key regulatory and pro-inflammatory cytokines, which are known to be important the immune response to UTI, including Interleukin (IL)-1β, IL-12(p40), and Monocyte Chemoattractant Protein-1 (MCP-1). Finally, we demonstrate that other cytokines, including IL-17 are induced significantly in the S. agalactiae-infected bladder regardless of age and parity status. Collectively, these findings show that the host environment plays an important role in influencing the severity of S. agalactiae UTI; infection dynamics, particularly in the context of bacteriuria, depend on age and parity, which also affect the nature of innate immune responses to infection., Competing Interests: The authors have declared that no competing interests exist.

In this study, we retrospectively reviewed the clinical experience of patients receiving doripenem-containing regimens for the treatment of healthcare-associated infections (HCAIs) in a tertiary care center and assessed the clinical usefulness of doripenem therapy in this clinical setting. In this retrospective study, the medical records of all adult patients who had ever received doripenem-containing therapy for the treatment of HCAIs were reviewed between September 1, 2012 and August 31, 2014, and the following data were extracted: age, gender, type of infection, disease severity, underlying comorbidities or conditions, and laboratory results. Additionally, we also extracted data regarding the rates of mortality and clinical and microbiological response. A total of 184 adult patients with HCAIs who had received doripenem-containing therapy were included in this study. Respiratory tract infections (n = 91, 49.5%) were the most common type of infection, followed by urinary tract infections, intra-abdominal infections and skin and soft tissue infections. The mean APACHE II score was 14.5. The rate of clinical success was 78.2%, and the overall in-hospital mortality rate was only 13.0%. Among patients, in-hospital mortality was independently and significantly associated with APACHE II score (odds ratio (OR), 1.2825; 95% CI, 1.1123-1.4788) and achieving clinical success (OR, 0.003; 95% CI, 0.0003-0.409). In conclusion, the overall in-hospital mortality rate was low and the clinical success rate was high among HCAI patients receiving doripenem treatment. These results suggest that doripenem may be judiciously used for the treatment of patients with HCAIs., Competing Interests: The authors have declared that no competing interests exist.

Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified Regenerating islet-derived 3 gamma (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic Escherichia coli (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC in vitro, but does kill Staphylococcus saprophyticus in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.

The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic Escherichia coli commonly possess the dsdCXA genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a dsdA mutant outcompetes wild type in the murine model of urinary tract infection. This "hypercolonization" was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of dsdA does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in rpoS and that some D-serine specific effects described in other studies may be affected by the rpoS status of the strains used. Inactivation of dsdA in the original clinical isolate of CFT073 gives CFT073 ΔdsdA a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 ΔdsdA is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic E. coli to colonize the murine urinary tract.

Urinary tract infections (UTIs) afflict over 9 million women in America every year, often necessitating long-term prophylactic antibiotics. One risk factor for UTI is frequent sexual intercourse, which dramatically increases the risk of UTI. The mechanism behind this increased risk is unknown; however, bacteriuria increases immediately after sexual intercourse episodes, suggesting that physical manipulation introduces periurethral flora into the urinary tract. In this paper, we investigated whether superinfection (repeat introduction of bacteria) resulted in increased risk of severe UTI, manifesting as persistent bacteriuria, high titer bladder bacterial burdens and chronic inflammation, an outcome referred to as chronic cystitis. Chronic cystitis represents unchecked luminal bacterial replication and is defined histologically by urothelial hyperplasia and submucosal lymphoid aggregates, a histological pattern similar to that seen in humans suffering chronic UTI. C57BL/6J mice are resistant to chronic cystitis after a single infection; however, they developed persistent bacteriuria and chronic cystitis when superinfected 24 hours apart. Elevated levels of interleukin-6 (IL-6), keratinocyte cytokine (KC/CXCL1), and granulocyte colony-stimulating factor (G-CSF) in the serum of C57BL/6J mice prior to the second infection predicted the development of chronic cystitis. These same cytokines have been found to precede chronic cystitis in singly infected C3H/HeN mice. Furthermore, inoculating C3H/HeN mice twice within a six-hour period doubled the proportion of mice that developed chronic cystitis. Intracellular bacterial replication, regulated hemolysin (HlyA) expression, and caspase 1/11 activation were essential for this increase. Microarrays conducted at four weeks post inoculation in both mouse strains revealed upregulation of IL-1 and antimicrobial peptides during chronic cystitis. These data suggest a mechanism by which caspase-1/11 activation and IL-1 secretion could predispose certain women to recurrent UTI after frequent intercourse, a predisposition predictable by several serum biomarkers in two murine models.

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

Objective: This study was designed to investigate whether increased urothelial cell apoptosis and chronic inflammation might contribute to recurrent urinary tract infection (UTI) in women., Methods: The bladder biopsy specimens were collected from thirty women with recurrent UTI and ten controls. The bladder biopsies were performed at one to two months after UTI episode had been completely resolved and urine analysis and urine culture all showed negative. Immunofluorescence staining of the adhesive protein E-cadherin, mast cell and TUNEL were performed in all the bladder specimens. In addition, western blots were also performed to analyze the inflammatory proteins (phospho-p38, tryptase) and apoptotic protein (Bax) in the bladder mucosa specimens between patients with recurrent UTI and controls., Results: Immunofluorescence staining showed significantly lower E-cadherin in the recurrent UTI bladder tissue compared with the controls (25.4±8.9 v 42.4±16.7, p<0.0001). The mast cell expression was significantly stronger in the recurrent UTI bladder tissue compared with the controls (2.5±1.8 v 1.3±1.2, p = 0.046). TUNEL staining revealed a significantly higher numbers of apoptotic cells in the recurrent UTI bladder tissue compared with the control bladder tissue (1.5±1.8 v 0.08±0.3, p<0.0001). Western blot analysis also showed that the expressions of tryptase and Bax increased in five recurrent UTI specimens compared with two normal control specimens., Conclusion: Chronic inflammation, urothelial cell apoptosis and impairment of barrier function of urothelial cells might contribute to recurrent UTI in women.

Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20-80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organs.

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract.

Schistosoma haematobium is the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. While humans readily develop urogenital schistosomiasis, the lack of an experimentally-tractable model has greatly impaired our understanding of the mechanisms that underlie this important disease. We have developed an improved mouse model of S. haematobium urinary tract infection that recapitulates several aspects of human urogenital schistosomiasis. Following microinjection of purified S. haematobium eggs into the bladder wall, mice consistently develop macrophage-rich granulomata that persist for at least 3 months and pass eggs in their urine. Importantly, egg-injected mice also develop urinary tract fibrosis, bladder dysfunction, and various urothelial changes morphologically reminiscent of human urogenital schistosomiasis. As expected, S. haematobium egg-induced immune responses in the immediate microenvironment, draining lymph nodes, and systemic circulation are associated with a Type 2-dominant inflammatory response, characterized by high levels of interleukin-4, eosinophils, and IgE. Taken together, our novel mouse model may help facilitate a better understanding of the unique pathophysiological mechanisms of epithelial dysfunction, tissue fibrosis, and oncogenesis associated with urogenital schistosomiasis.

Chronic infections are an increasing problem due to the aging population and the increase in antibiotic resistant organisms. Therefore, understanding the host-pathogen interactions that result in chronic infection is of great importance. Here, we investigate the molecular basis of chronic bacterial cystitis. We establish that introduction of uropathogenic E. coli (UPEC) into the bladders of C3H mice results in two distinct disease outcomes: resolution of acute infection or development of chronic cystitis lasting months. The incidence of chronic cystitis is both host strain and infectious dose-dependent. Further, development of chronic cystitis is preceded by biomarkers of local and systemic acute inflammation at 24 hours post-infection, including severe pyuria and bladder inflammation with mucosal injury, and a distinct serum cytokine signature consisting of elevated IL-5, IL-6, G-CSF, and the IL-8 analog KC. Mice deficient in TLR4 signaling or lymphocytes lack these innate responses and are resistant, to varying degrees, to developing chronic cystitis. Treatment of C3H mice with the glucocorticoid anti-inflammatory drug dexamethasone prior to UPEC infection also suppresses the development of chronic cystitis. Finally, individuals with a history of chronic cystitis, lasting at least 14 days, are significantly more susceptible to redeveloping severe, chronic cystitis upon bacterial challenge. Thus, we have discovered that the development of chronic cystitis in C3H mice by UPEC is facilitated by severe acute inflammatory responses early in infection, which subsequently are predisposing to recurrent cystitis, an insidious problem in women. Overall, these results have significant implications for our understanding of how early host-pathogen interactions at the mucosal surface determines the fate of disease.

Background: Polymorphisms affecting Toll-like receptor (TLR) structure appear to be rare, as would be expected due to their essential coordinator role in innate immunity. Here, we assess variation in TLR4 expression, rather than structure, as a mechanism to diversify innate immune responses., Methodology/principal Findings: We sequenced the TLR4 promoter (4,3 kb) in Swedish blood donors. Since TLR4 plays a vital role in susceptibility to urinary tract infection (UTI), promoter sequences were obtained from children with mild or severe disease. We performed a case-control study of pediatric patients with asymptomatic bacteriuria (ABU) or those prone to recurrent acute pyelonephritis (APN). Promoter activity of the single SNPs or multiple allelic changes corresponding to the genotype patterns (GPs) was tested. We then conducted a replication study in an independent cohort of adult patients with a history of childhood APN. Last, in vivo effects of the different GPs were examined after therapeutic intravesical inoculation of 19 patients with Escherichia coli 83972. We identified in total eight TLR4 promoter sequence variants in the Swedish control population, forming 19 haplotypes and 29 genotype patterns, some with effects on promoter activity. Compared to symptomatic patients and healthy controls, ABU patients had fewer genotype patterns, and their promoter sequence variants reduced TLR4 expression in response to infection. The ABU associated GPs also reduced innate immune responses in patients who were subjected to therapeutic urinary E. coli tract inoculation., Conclusions: The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.

Background: Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). While UTIs are typically considered extracellular infections, it has been recently demonstrated that UPEC bind to, invade, and replicate within the murine bladder urothelium to form intracellular bacterial communities (IBCs). These IBCs dissociate and bacteria flux out of bladder facet cells, some with filamentous morphology, and ultimately establish quiescent intracellular reservoirs that can seed recurrent infection. This IBC pathogenic cycle has not yet been investigated in humans. In this study we sought to determine whether evidence of an IBC pathway could be found in urine specimens from women with acute UTI., Methods and Findings: We collected midstream, clean-catch urine specimens from 80 young healthy women with acute uncomplicated cystitis and 20 asymptomatic women with a history of UTI. Investigators were blinded to culture results and clinical history. Samples were analyzed by light microscopy, immunofluorescence, and electron microscopy for evidence of exfoliated IBCs and filamentous bacteria. Evidence of IBCs was found in 14 of 80 (18%) urines from women with UTI. Filamentous bacteria were found in 33 of 80 (41%) urines from women with UTI. None of the 20 urines from the asymptomatic comparative group showed evidence of IBCs or filaments. Filamentous bacteria were present in all 14 of the urines with IBCs compared to 19 (29%) of 66 samples with no evidence of IBCs (p < 0.001). Of 65 urines from patients with E. coli infections, 14 (22%) had evidence of IBCs and 29 (45%) had filamentous bacteria, while none of the gram-positive infections had IBCs or filamentous bacteria., Conclusions: The presence of exfoliated IBCs and filamentous bacteria in the urines of women with acute cystitis suggests that the IBC pathogenic pathway characterized in the murine model may occur in humans. The findings support the occurrence of an intracellular bacterial niche in some women with cystitis that may have important implications for UTI recurrence and treatment.