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Background: Cathelicidin is a proposed defender against infection of the urinary tract via its antimicrobial properties, but its activity has not been delineated in a dedicated cystitis model., Methods: Female C57Bl/6 mice, wild type or deficient in cathelin-related antimicrobial peptide (CRAMP; an ortholog of the sole human cathelicidin, LL-37), were infected transurethrally with the cystitis-derived uropathogenic Escherichia coli (UPEC) strain UTI89. Infection course was evaluated by bladder titers, intracellular bacterial community quantification, and histological analysis. Immune responses and resolution were characterized through cytokine profiling, microscopy, and quantitation of epithelial recovery from exfoliation., Results: CRAMP-deficient mice exhibited significantly lower bladder bacterial loads and fewer intracellular bacterial communities during acute cystitis. Although differences in bacterial titers were evident as early as 1 hour after infection, CRAMP-deficient mice showed no baseline alterations in immune activation, uroepithelial structure, apical expression of uroplakins (which serve as bacterial receptors), or intracellular bacterial growth rate. CRAMP-deficient hosts demonstrated less intense cytokine responses, diminished neutrophil infiltration, and accelerated uroepithelial recovery., Conclusions: Mice lacking the antimicrobial peptide cathelicidin experienced less severe infection than wild-type mice in a well-established model of cystitis. Although CRAMP exhibits in vitro antibacterial activity against UPEC, it may enhance UPEC infection in the bladder by promoting epithelial receptivity and local inflammation., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Proteus mirabilis is a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms for P. mirabilis to establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulating mrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted from rpoE mutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness of P. mirabilis in the host, including the avoidance of immune attacks. Accordingly, rpoE mutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and the rpoE mutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression of rpoE by the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness of P. mirabilis, to build up a UTI., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Several rod-shaped pathogens including Escherichia coli, Salmonella spp. and Klebsiella pneumonia are capable of adopting highly filamentous cell shapes under certain circumstances. This phenomenon occurs as a result of continued cell elongation during growth without the usual septation into single rod-shaped cells. Evidence has emerged over the past decade suggesting that this morphological transformation is controlled and reversible and provides selective advantages under certain growth conditions, such as during infection in humans. In order to identify the factors which induce filamentation of bacterial pathogens and study the advantages of bacterial morphological plasticity, methods are needed to accurately quantify changes in bacterial cell shape. In this study, we present a method for quantification of bacterial filamentation based on automatic detection and measurement of bacterial units in focus-stacked microscopy images. Used in combination with a flow-chamber based in vitro cystitis model, we study the factors involved in filament formation by uropathogenic E. coli (UPEC) during infection. The influence of substratum surface, intracellular proliferation and flow media on UPEC filamentation is evaluated. We show that reversible UPEC filamentation during cystitis is not dependent on intracellular infection, which previous studies have suggested. Instead, we find that filamentation can be induced by contact with surfaces, both biological and artificial. Lastly our data indicate that UPEC filamentation is induced by trace-amounts of specific components in urine, rather than being a generic stress-response to high urine salt concentrations. The study shows that the combined methodology is generally useful for investigation of bacterial morphological transitions during cell infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Uropathogenic Escherichia coli (UPEC) is the most common agent of urinary tract infection (UTI). The classic model of pathogenesis proposes the ascent of UPEC by the urethra and external adherence to the urothelium. Recently, the ability of UPEC to invade urothelial cells and to form intracellular bacterial communities (IBCs) has been described., Methods: The objective of the present study was to determine the presence of intracellular bacteria (IB) in children with UTI caused by E. coli and to characterize its virulence attributes and its relation with clinical outcomes. One hundred thirty-three children with E. coli UTI who attended a reference children's hospital between June and November 2012 were included. Urine samples were analyzed by optical and confocal microscopy looking for exfoliated urothelial cells with IB. Phylogenetic group and 24 virulence factors of UPEC were determined using multiplex polymerase chain reaction. Medical records were analyzed., Results: The presence of IB was detected in 49 of 133 (36.8%) samples by confocal microscopy, in 30 cases as IBC, and in 19 as isolated intracellular bacteria (IIB). Only 50% of these cases could be detected by light microscopy. Seventy-four medical records were analyzed, 34 with IBC/IIB, 40 without IB. Any virulence gene was associated with IBC/IIB. The presence of IBC/IIB was associated with recurrent UTI (odds ratio [OR], 3.3; 95% confidence interval [CI], 1.3-9; P = .017), especially in children without urinary tract functional or morphological abnormalities (OR, 8.0; 95% CI, 2.3-27.4; P = .000). IBCs were associated with lower urinary tract syndrome (OR, 3.6; 95% CI, 1.1-11.8; P = .05) and absence of fever (P = .009)., Conclusions: IBCs/IIB could explain a high proportion of children with recurrent UTI., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has a broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7, but regulation of its antimicrobial activity has not been well defined. Here, we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on the antimicrobial activity of RNase 7. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time polymerase chain reaction showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.

Bacillus Calmette-Guérin (BCG) has been used to treat non-muscle-invasive bladder cancer for more than 30 years. It is one of the most successful biotherapies for cancer in use. Despite long clinical experience with BCG, the mechanism of its therapeutic effect is still under investigation. Available evidence suggests that urothelial cells (including bladder cancer cells themselves) and cells of the immune system both have crucial roles in the therapeutic antitumour effect of BCG. The possible involvement of bladder cancer cells includes attachment and internalization of BCG, secretion of cytokines and chemokines, and presentation of BCG and/or cancer cell antigens to cells of the immune system. Immune system cell subsets that have potential roles in BCG therapy include CD4(+) and CD8(+) lymphocytes, natural killer cells, granulocytes, macrophages, and dendritic cells. Bladder cancer cells are killed through direct cytotoxicity by these cells, by secretion of soluble factors such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), and, to some degree, by the direct action of BCG. Several gaps still exist in our knowledge that should be addressed in future efforts to understand this biotherapy of cancer.

Despite considerable controversy about their effects, cranberries in various forms have been used widely for several decades to prevent as well as treat urinary tract infections (UTIs). The purpose of this article is to present a review of research-based information regarding the ability of cranberries to prevent UTIs in adults at risk for UTIs. Current evidence suggests that cranberries decrease bacterial adherence to uroepithelial cells and thus decrease the incidence of UTIs without adverse effects in most individuals. Thus clinicians may safely advise patients that cranberries are helpful in preventing UTIs. Cranberries may be a viable adjunct to antibiotics for patients with repeated UTIs.

We have previously demonstrated that the C. albicans pre-vacuolar protein sorting gene VPS4 is required for extracellular secretion of the secreted aspartyl proteases Sap2p and Saps4-6p. Furthermore, the vps4Δ null mutant has been shown to be markedly hypovirulent in a murine tail vein model of disseminated candidiasis. In these experiments, we sought to further define the role of the pre-vacuolar secretion pathway mediated by the pre-vacuolar sorting gene VPS4 in the pathogenesis of epithelial and mucosal infection using a broad range of virulence models. The C. albicans vps4Δ mutant demonstrates reduced tolerance of cell wall stresses compared to its isogenic, complemented control strain. In an in vitro oral epithelial model (OEM) of tissue invasion, the vps4Δ mutant caused reduced tissue damage compared to controls. Further, the vps4Δ mutant was defective in macrophage killing in vitro, and was attenuated in virulence in an in vivo Caenorhabditis elegans model representative of intestinal epithelial infection. In contrast, the vps4Δ mutant caused a similar degree of tissue damage in an in vitro uroepithelial model of Candida infection compared with controls. Furthermore, in an in vivo murine model of vaginal candidiasis there was no reduction in fungal colony burden and no differences in vaginal histopathology compared to wild-type and complemented controls. These results suggest that VPS4 contributes to several key aspects of oral epithelial but not uroepithelial infection, and in contrast to systemic infection, plays no major role in the pathogenesis of Candida vaginitis. By using a wide range of virulence models, we demonstrate that C. albicans VPS4 contributes to virulence according to the specific tissue that is infected. Thus, in order to gain a full understanding of C. albicans virulence in relation to a particular gene or pathway of interest, a selected range of infection models may need to be utilized.

Clinical data suggest an impact of estrogen on the pathogenesis of urinary tract infections (UTI). In particular, women after menopause often suffer from recurrent UTI, characterized by at least three acute UTI episodes within a year. Aside from bacterial factors promoting persistence within the urinary bladder, the low estrogen levels induce structural and chemical changes in the urogenital tract which facilitate UTI. Increased residual urine volume and changes in the vaginal microflora are well documented risk factors. Local supplementation with estrogen can at least partly reverse these changes. Treatment allows the re-establishment of a lactobacilli-dominated vaginal microflora and improves epithelial differentiation and integrity in the urogenital tract. This estrogenic effect on the epithelium is marked by an increased production of antimicrobial peptides and a tighter intercellular connection, preventing bacteria from reaching cells where they can hide and later emerge and cause a new infection. Estrogen in the dosages and applications used to date is considered safe for the endometrium in the majority of women. Based on the actions and safety of estrogen, local supplementation thus offers a treatment option for postmenopausal women suffering from recurrent UTI., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Bacterial urinary tract infections (UTI) are a major growing concern worldwide. Uropathogenic Escherichia coli has been shown to invade the urothelium during acute UTI in mice and humans, forming intracellular reservoirs that can evade antibiotics and the immune response, allowing recurrence at a later date. Other bacterial species, such as Staphylococcus saprophyticus, Klebsiella pneumonia and Salmonella enterica have also been shown to be invasive in acute UTI. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the elderly population, is poorly understood. Moreover, the species of bacteria involved remains largely unknown. A previous study of a large cohort of non-acute LUTS patients found that Enterococcus faecalis was frequently found in urine specimens. E. faecalis accounts for a significant proportion of chronic bladder infections worldwide, although the invasive lifestyle of this uropathogen has yet to be reported. Here, we wanted to explore this question in more detail. We harvested urothelial cells shed in response to inflammation and, using advanced imaging techniques, inspected them for signs of bacterial pathology and invasion. We found strong evidence of intracellular E. faecalis harboured within urothelial cells shed from the bladder of LUTS patients. Furthermore, using a culture model system, these patient-isolated strains of E. faecalis were able to invade a transitional carcinoma cell line. In contrast, we found no evidence of cellular invasion by E. coli in the patient cells or the culture model system. Our data show that E. faecalis is highly competent to invade in this context; therefore, these results have implications for both the diagnosis and treatment of chronic LUTS.

Ethnopharmacological Relevance: Traditional usage suggests Citrus reticulata Blanco seeds have beneficial effects against infection. The purpose of this study was to investigate the effect of Citrus reticulata on the uroepithelium and to determine the mechanisms responsible for protection against urinary tract infection (UTI)., Materials and Methods: Human bladder cell lines T24 and 5637 were employed in a cell culture infection model to determine the effects of Citrus reticulata treatment on Escherichia coli adherence and invasion of the uroepithelium. β1 integrin and caveolin-1 mRNA expression was assessed using RT real-time PCR. β1 integrin protein expression was confirmed by Western Blot. The effect of Citrus reticulata on bacteria was investigated using antibacterial sensitivity, yeast agglutination and biofilm assays., Results: Citrus reticulata treatment decreased β1 integrin expression and reduced bacterial invasion while adhesion of uroepithelial cells was not affected. Caveolin-1 expression was not influenced either and Citrus reticulata did neither exhibit any direct antimicrobial effect nor interfered with type 1 fimbriae binding., Conclusions: Our results show that Citrus reticulata has a protective effect on the uroepithelium as seen by reduced bacterial invasion of uroepithelial cells. These properties suggest that seeds from Citrus reticulata may have therapeutic potential in preventing UTI., (© 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: In order to define the virulence factors between Chinese herb-resistant uropathogenic E. coli and susceptible strains, the UPEC isolates were classified into two groups according to its resistance to Chinese herbs., Materials and Methods: The susceptibility profile of strains was determined by disk diffusion method. PCR systems were used to detect genes encoding papC, Aer, hly and cnf1. Isolated human urothelial cells were incubated in vitro and investigated with light microscope immunohistochemistry. Adhesion of E. coli to urothelial cells was studied in vitro., Results: The results showed that, among the 105 UPEC isolates, 18 were resistant to the herbal concoction. Cnf1 and papC occurred in ≥66.7%, of herb-resistant isolates, while, hly and Aer occurred in 22.2% and 27.8% of strains respectively. Only one gene (Cnf1) occurred in >40%, of Herb-susceptible isolates. Other genes were also found in susceptible isolates: papC (20.7%), hly (11.5%), and Aer (6.9%). Light microscopy and immunochemical investigations demonstrated the normal pelvic transitional epithelial cells cultured. The adherence of strains in both groups increased in 30 min., and reached its peak at 60, (Susceptible E. coli) or 120 min., (Resistant E. coli). The adhesion of the susceptible bacteria to human uroepithelial cells was significantly lower compared with that of the resistant E. coli (p<0.05)., Conclusion: These findings revealed that, Chinese herb-resistant uropathogenic E. coli isolates that are hemolytic, and have Aer, papC, hly, Cnf1 genes are more able to be uropathogenic and adherent.

A mouse model of cystitis caused by uropathogenic Escherichia coli was used to study the distribution of gallium in bladder tissue following oral administration of gallium maltolate during urinary tract infection. The median concentration of gallium in homogenized bladder tissue from infected mice was 1.93 μg/g after daily administration of gallium maltolate for 5 days. Synchrotron X-ray fluorescence imaging and X-ray absorption spectroscopy of bladder sections confirmed that gallium arrived at the transitional epithelium, a potential site of uropathogenic E. coli infection. Gallium and iron were similarly but not identically distributed in the tissues, suggesting that at least some distribution mechanisms are not common between the two elements. The results of this study indicate that gallium maltolate may be a suitable candidate for further development as a novel antimicrobial therapy for urinary tract infections caused by uropathogenic E. coli.

Urinary tract infections (UTIs) represent a common and quite costly medical problem, primarily affecting the female population which may be due to a shorter urethra. The bacterium Escherichia coli are mainly responsible for most uncomplicated UTIs. Cranberry antibacterial effects have widely been studied in vitro, and laboratory and clinical studies have also been performed to elucidate the mechanisms of cranberry actions and the clinical benefits of cranberry consumption against UTIs. The present review aimed to summarize the proposed mechanisms of cranberry actions against UTIs and the clinical trials that evaluated the efficacy of supplementing cranberry products in different subpopulations. Taking into consideration the existing data, cranberry consumption may prevent bacterial adherence to uroepithelial cells which reduces the development of UTI. Cranberry consumption could also decreasing UTI related symptoms by suppressing inflammatory cascades as an immunologic response to bacteria invasion. The existing clinical trials suggest that the beneficial effects of cranberry against UTIs seem to be prophylactic by preventing the development of infections; however, they exert low effectiveness in populations at increased risk for contracting UTIs. Additional well-designed, double-blind, placebo-controlled clinical trials that use standardized cranberry products are strongly justified in order to determine the efficiency of cranberry on the prevention of UTIs in susceptible populations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

Epidemiological data imply a role of estrogen in the pathogenesis of urinary tract infections (UTIs), although the underlying mechanisms are not well understood. However, it is thought that estrogen supplementation after menopause decreases the risk of recurrent infections. We sought to investigate the influence of estrogen on host-pathogen interactions and the consequences for UTI pathogenesis. We analyzed urothelial cells from menstruating and postmenopausal women before and after a 2-week period of estrogen supplementation, and also studied the influence of estradiol during Escherichia coli UTI in a mouse infection model. Important findings were confirmed in two human urothelial cell lines. We identified two epithelial defense mechanisms modulated by estrogen. Estrogen induced the expression of antimicrobial peptides, thereby enhancing the antimicrobial capacity of the urothelium and restricting bacterial multiplication. In addition, estrogen promoted the expression and redistribution of cell-cell contact-associated proteins, thereby strengthening the epithelial integrity and preventing excessive loss of superficial cells during infection. These two effects together may prevent bacteria from reaching deeper layers of the urinary tract epithelium and developing reservoirs that can serve as a source for recurrent infections. Thus, this study presents some underlying mechanisms for the beneficial effect of estradiol after menopause and supports the application of estrogen in postmenopausal women suffering from recurrent UTI.

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.

Adhesion of bacteria to epithelial tissue is an essential step in the progression of the urinary tract infections. Reduction of virulence factors responsible for microbial attachment may help to decrease or inhibit colonization of the host organism by pathogens. In the age of increasing bacterial antibiotic resistance, more and more attention is being paid to the use of plants and/or their bioactive components in the prevention and treatment of human infections. Asiatic acid (AA) and ursolic acid (UA), two plant secondary metabolites, were used as potential antibacterial agents. The current study aimed to determine the possible impact of AA and UA on morphology, hydrophobicity, and adhesion of clinical uropathogenic Escherichia coli strains (UPEC) to the uroepithelial cells. Our work describes for the first time the effects exerted by AA and UA on virulence factors of UPECs. The impact of both acids on the cell surface hydrophobicity of the investigated strains was very weak. The results clearly show the influence of AA and UA on the presence of P fimbriae and curli fibers, morphology of the UPECs cells and their adhesion to epithelium; however, some differences between activities of AA and UA were found.

Quorum sensing (QS) plays a pivotal role in the virulence of Pseudomonas aeruginosa. Urinary tract infections (UTI's) caused by P. aeruginosa leads to tissue destruction and inflammation. Interaction of host immune cells and pathogen at the epithelial surface are regulated by cytokines. Role of virulence factors has been implicated in the induction of renal cytokine responses. However, information regarding the role of QS, an important virulence factor of P. aeruginosa, is lacking. This study examined role of QS signal molecules for their ability to induce pro and anti-inflammatory renal cytokines and inflammation in experimental UTI. For this, both standard parent P. aeruginosa strain (PAO1) capable of producing QS signal molecules and its isogenic single and double mutant strains lacking this ability were used. Mice infected with QS producer strain PAO1, evoked significant renal tissue inflammation and cytokine response with high neutrophil migration and AHL production in vivo. Mild inflammation, low level of renal cytokines and other factors were observed in mice infected with QS single and double mutant strains. These results provide first evidence regarding the role of QS signal molecules and point towards the importance of AHLs in induction of renal cytokines and tissue inflammatory responses., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Since the first report in 1976, accumulated clinical evidence has supported intravesical Bacillus Calmette-Guerin (BCG) therapy as one of the standard methods of management of intermediate- and high-risk non-muscle invasive bladder cancer. Despite its efficacy, intravesical BCG therapy is associated with a variety of adverse events (AEs), most of which are tolerable or controllable with supportive care. However, some patients receiving intravesical BCG therapy may experience uncommon but severe AEs, leading to cessation of BCG therapy. Not all, but most severe AEs result from either local or systemic infection with live BCG. Intravesical instillation of BCG elicits multiple immune reactions, although the precise immunological mechanism of BCG therapy is not clear. It is convenient to separate the complex reactions into the following three categories: infection of urothelial cells or bladder cancer cells, induction of immune reactions, and induction of antitumor effects. Recently, our knowledge about each category has increased. Based on this understanding, predictors of the efficacy of intravesical BCG therapy, such as urinary cytokine measurement and cytokine gene polymorphism, have been investigated. Recently, preclinical studies using a novel engineered mycobacterium vaccine have been conducted to overcome the limitations of BCG therapy. One approach is Th1 cytokine-expressing recombinant forms of BCG; another approach is development of non-live bacterial agents to avoid AEs due to live BCG infection. We also briefly describe our approach using an octaarginine-modified liposome-incorporating BCG cell wall component to develop future substitutes for live BCG., (© 2012 Japanese Cancer Association.)

The adherence of uropathogenic Escherichia coli (UPEC) to the host urothelial surface is the first step for establishing UPEC infection. Uroplakin Ia (UPIa), a glycoprotein expressed on bladder urothelium, serves as a receptor for FimH, a lectin located at bacterial pili, and their interaction initiates UPEC infection. Surfactant protein D (SP-D) is known to be expressed on mucosal surfaces in various tissues besides the lung. However, the functions of SP-D in the non-pulmonary tissues are poorly understood. The purposes of this study were to investigate the possible function of SP-D expressed in the bladder urothelium and the mechanisms by which SP-D functions. SP-D was expressed in human bladder mucosa, and its mRNA was increased in the bladder of the UPEC infection model in mice. SP-D directly bound to UPEC and strongly agglutinated them in a Ca(2+)-dependent manner. Co-incubation of SP-D with UPEC decreased the bacterial adherence to 5637 cells, the human bladder cell line, and the UPEC-induced cytotoxicity. In addition, preincubation of SP-D with 5637 cells resulted in the decreased adherence of UPEC to the cells and in a reduced number of cells injured by UPEC. SP-D directly bound to UPIa and competed with FimH for UPIa binding. Consistent with the in vitro data, the exogenous administration of SP-D inhibited UPEC adherence to the bladder and dampened UPEC-induced inflammation in mice. These results support the conclusion that SP-D can protect the bladder urothelium against UPEC infection and suggest a possible function of SP-D in urinary tract.

Modulation of the immune response is an established feature of certain lactobacilli. CXCL8 is an inflammatory chemokine released by the urinary tract mucosa after contact with uropathogenic Escherichia coli during urinary tract infection and is crucial for proper infiltration of immune cells. Nevertheless, persistently high levels of CXCL8 are associated with pathogenicity and malignancy. In this study, we tested twelve Lactobacillus strains for their ability to influence CXCL8 release from urothelial cells. We evaluated how strains from different Lactobacillus species could regulate CXCL8 in human 5637 urothelial cells, either resting cells or cells concomitantly challenged with heat-killed E. coli. A majority of the tested species altered CXCL8 release from the urothelial cells after 24 hours of stimulation. Most species increased CXCL8 release, whereas a few lactobacilli efficiently suppressed CXCL8 secretion from E. coli-challenged cells. While strong CXCL8 modulators such as Lactobacillus reuteri and Lactobacillus delbrueckii were unable to degrade CXCL8 in the extracellular environment, effects on IL8 transcription were evident for selected lactobacilli. Although IL8 transcription was affected by lactobacilli, the influence on mRNA transcript did not correlate to the impact on CXCL8 release. Phylogenetic analysis based on a 16S rRNA dendrogram of the tested lactobacilli and their effect on CXCL8 revealed some linkage to specific Lactobacillus groups. Testing the immunomodulatory nature of lactobacilli can prove important when selecting new probiotic microbes. Moreover, we believe that phylogenetic and phenotypic similarities could be used to analyse the traits governing such modulation.

The urinary bladder is a common site of bacterial infection with a majority of cases attributed to uropathogenic Escherichia coli. Sequelae of urinary tract infections (UTIs) include the loss of urothelial barrier function and subsequent clinical morbidity secondary to the permeation of urine potassium, urea and ammonia into the subepithelium. To date there has been limited research describing the mechanism by which this urothelial permeability defect develops. The present study models acute uropathogenic E. coli infection in vitro using intact canine bladder mucosa mounted in Ussing chambers to determine whether infection induces primarily a transcellular or paracellular permeability defect. The Ussing chamber sustains tissue viability while physically separating submucosal and lumen influences, so this model is ideal for quantitative measurement of transepithelial electrical resistance (TER) to assess alterations of urothelial barrier function. Using this model, changes in both tissue ultrastructure and TER indicated that uropathogenic E. coli infection promotes a paracellular permeability defect associated with the failure of umbrella cell tight junction formation and umbrella cell sloughing. In addition, bacterial interaction with the urothelium promoted secretion of cytokines from the urinary bladder with bioactivity capable of modulating epithelial barrier function including tumour necrosis factor-α, interleukin (IL)-6 and IL-15. IL-15 secretion by the infected bladder mucosa is a novel finding and, because IL-15 plays key roles in reconstitution of tight junction function in damaged intestine, this study points to a potential role for IL-15 in UTI-induced urothelial injury., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multidrug-resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic Escherichia coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity, and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the quiescent intracellular reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection, QIR, ASB, or chronic cystitis, is determined within the first 24 h of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Cranberry extract has been reported as a therapeutic agent, mainly in urinary tract infections due to its anti-adhesive capacity. In order to compare the effects of proanthocyanidin (procyanidin) (PAC)-standardized cranberry extracts and commercial PAC A2, we first investigated the presence of genes encoding known adhesins on 13 strains of uropathogenic strains coming from patients with cystisis. After this characterization, the anti-adhesive effects of PAC A2 were assayed on selected uropathogenic Escherichia coli strains before testing cranberry extracts. Before checking inhibitory effect on bacterial adhesion to cells, we showed that neither PAC A2 or three cranberry extracts (A, B, and C) specifically inhibited the growth and did not supply any potential nutrient to E. coli strains, including the unrelated control strain. PAC A2 exhibited an inhibitory effect on the adhesion of two selected uropathogenic strains of E. coli. This work also showed that a preliminary exposure of bacteria to PAC A2 significantly reduced the adhesion. This phenomenon has been also observed with a lesser impact when uroepithelial cells were pretreated with PAC A2. Moreover, the assays were more robust when bacteria were in fast growing conditions (exponential phase): the adhesion to uroepithelial cells was greater. Significant reduction of adhesion to urepithelial cells was observed: around 80% of inhibition of adhesion with the cranberry extracts at equivalent PAC concentration of 50 μg/mL. The effects of the different assayed extracts were not obviously different except for extract B, which inhibited approximately 55% of adhesion at an equivalent PAC concentration of 5 μg/mL.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

Liquid-based urine cytology (LB-URC) was evaluated for cytological diagnosis and detection of human papillomavirus (HPV), Mycoplasma, and Ureaplasma. Midstream urine samples were collected from 141 male patients with urethritis and 154 controls without urethritis, and sediment cells were preserved in liquid-based cytology solution. Urethral swabs from urethritis patients were tested for the presence of Neisseria gonorrhoeae and Chlamydia trachomatis. Papanicolaou tests were performed for cytological evaluation. HPV, Mycoplasma, and Ureaplasma genomes were determined by PCR-based methods, and localization of HPV DNA in urothelial cells was examined by in situ hybridization (ISH). The β-globin gene was positive in 97.9% of LB-URC samples from urethritis patients and in 97.4% of control samples, suggesting that high-quality cellular DNA was obtained from the LB-URC samples. HPV DNA was detected in 29 (21.0%) urethritis cases and in five (3.3%) controls (P < 0.05). HPV type 16 (HPV 16) was most commonly found in urethritis patients. Cytological evaluations could be performed for 92.1% of urethritis patients and 64.3% of controls. Morphological changes suggestive of HPV infection were seen in 20.7% of the HPV-positive samples, and ISH demonstrated the presence of HPV DNA in both squamous and urothelial cells in HPV-positive samples. Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were detected in 14.5%, 10.9%, 6.5%, and 12.3% of urethritis patients, respectively. The prevalence rates of these microorganisms (except Ureaplasma parvum) were significantly higher in urethritis cases than controls (P < 0.05). LB-URC is applicable for detection of HPV, Mycoplasma, and Ureaplasma. HPV infection occurs in urothelial cells, especially in gonococcal urethritis.

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10-deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1(+) vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Ethnopharmacological Relevance: Labisia pumila var. alata (LPva) is a traditional medicinal herb used by Malaysian women to treat many ailments of the genitourinary tract. Its phytoestrogenic properties suggest potential to prevent recurrent urinary tract infection (UTI) in women post menopause. The aim of this study was therefore to investigate the mechanisms of action of LPva in an in vitro model of UTI., Materials and Methods: Bladder epithelial cell lines T24 and 5637 and uropathogenic Escherichia coli (UPEC) strain CFT073 were used to model uroepithelial infection. The ability of LPva to induce programmed cell death was tested using the Annexin-V-FLUOS and TUNEL assays. Expression of caveolin-1, β1 integrin and antimicrobial peptides HBD-2 and LL-37 in response to LPva treatment and/or infection, was assessed using RT real-time PCR. Effects on protein expression were confirmed by Western blot analysis. Sensitivity and yeast agglutination assays were employed to determine if LPva had antimicrobial activities and/or interacted with type 1 fimbriae, respectively. Finally, bacterial adherence and invasion to cells treated with LPva was examined., Results: LPva induced uroepithelial apoptosis which was coupled with upregulated expression of caveolin-1 and downregulation of β1 integrin. LPva did not exhibit direct antimicrobial properties and did not influence antimicrobial peptide levels in cells. Additionally, LPva did not interact with type 1 fimbriae and did not affect adherence in comparison to non-treated control cells. However, LPva significantly reduced the number of intracellular UPEC in bladder epithelial cells., Conclusions: Our findings suggest that LPva has beneficial applications against UPEC infection due to its ability to induce programmed cell death and reduce bacterial invasion of the uroepithelium., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Neisseria gonorrhoeae, a sexually-transmitted gram-negative bacterium, causes gonorrhoea in humans. The min genes of N. gonorrhoeae are involved in cell division site selection with oxyR co-transcribed with these genes. The mutation in min genes and oxy R cause aberrant cell morphology and aggregation patterns, respectively. Our objective was to assess the contribution of neisserial min operon cell division genes i.e. minC, minD and oxyR in virulence. Compared to the N. gonorrhoeae parental strain (Ng CH811Str(R)), its isogenic mutants with insertionally inactivated minC (Ng CSRC1), minD (Ng CJSD1) or oxyR (Ng KB1) showed reduced adherence to and invasion of urethral epithelial cells. This may be explained by defective microcolony formation in the mutant strains, possibly owing to abnormal morphology and aggregation. The expression levels of surface virulence factors like Opa, pilin and lipooligosaccharide in the mutants were unchanged relative to Ng CH811Str(R). Furthermore, in urethral epithelial cells, the min and oxyR mutants induced the release of proinflammatory cytokines like IL6 and IL8 to levels similar to that induced by the parental strain. Taken together, our studies indicate that inactivation of minC, minD or oxyR in N. gonorrhoeae attenuates its ability to bind to and invade urethral epithelial cells without altering its potential to induce IL6 and IL8 release., (Copyright © 2011 Institut Pasteur. All rights reserved.)

Ethnopharmacological Relevance: Uropathogenic Escherichia coli is the major cause for urinary tract infections (UTI). Due to emerging antimicrobial resistances treatment of UTI is becoming increasingly difficult. Therefore, alternative treatment strategies are required. We sought to investigate the molecular mechanisms of a traditionally used decoction from Vietnamese dandelion (Lactuca indica L.) mediating local protection of the bladder epithelium., Materials and Methods: To study the influence of herbal preparations on the interaction between bladder epithelial cells and uropthogenic Escherichia coli, a cell culture model of UTI was applied. In addition, the direct effect of the herbal extract on Escherichia coli was analyzed., Results: Although no direct antibacterial activity was determined, Lactuca indica decreased bacterial colonization of bladder epithelial cells remarkably. Reduced bacterial adherence was accompanied by lowered activation of focal adhesion kinase (FAK) in Lactuca indica exposed cells. Treatment with a chemical FAK inhibitor supported this mechanism of action., Conclusions: These findings indicate that, in addition to its diuretic action, Lactuca indica exhibits secondary effects directly on epithelial cells which may protect against Escherichia coli infection. These properties might be useful for the development of alternative strategies in the treatment of UTI., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria.

Aim: Evaluation of influence of microbial epithelial interactions on growth properties and antagonistic activity of dominant and associated vaginal microflora., Materials and Methods: Growth characteristics and antagonistic activity changes were studied in associated (Staphylococcus aureus, Escherichia coli, Corynebacterium spp.) and dominant (Lactobacilus spp.) microflora during contact with vaginal epitheliocytes and influence of vaginal epitheliocyte secretory products by using the developed technique., Results: Secretory products of vaginal epitheliocytes and vaginal epitheliocytes differentially influence growth factors of vaginal microbiocenosis. Suppression of S. aureus and E. coli biomass growth based predominately on extracellular secretory products of epitheliocytes was observed. Stimulation of growth properties was prevalent for lactobacilli and corynebacteria and was observed in contact interaction as well as during effect of epitheliocyte extracellular secretory products., Conclusion: The resulting bacterial-epithelial interaction is a differentiating feature between autochthonous and allochthonous microflora due to the creation of selective advantages for dominant microsymbionts by growth properties and antagonistic activity stimulation, and biomass growth suppression of associated symbionts.

Scope: Atomic force microscopy (AFM) was used to directly measure the nanoscale adhesion forces between P-fimbriated Escherichia coli (E. coli) and human uroepithelial cells exposed to cranberry juice, in order to reveal the molecular mechanisms by which cranberry juice cocktail (CJC) affects bacterial adhesion., Methods and Results: Bacterial cell probes were created by attaching P-fimbriated E. coli HB101pDC1 or non-fimbriated E. coli HB101 to AFM tips, and the cellular probes were used to directly measure the adhesion forces between E. coli and uroepithelial cells in solutions containing: 0, 2.5, 5, 10, and 27 wt% CJC. Macroscale attachment of E. coli to uroepithelial cells was measured and correlated to nanoscale adhesion force measurements. The adhesion forces between E. coli HB101pDC1 and uroepithelial cells were dose-dependent, and decreased from 9.32±2.37 nN in the absence of CJC to 0.75±0.19 nN in 27 wt% CJC. Adhesion forces between E. coli HB101 and uroepithelial cells were low in buffer (0.74±0.18 nN), and did not change significantly in CJC (0.78±0.18 nN in 27 wt% CJC; P=0.794)., Conclusion: Our study shows that CJC significantly decreases nanoscale adhesion forces between P-fimbriated E. coli and uroepithelial cells., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Numerous antibiotics have proven to be effective at ameliorating the clinical symptoms of urinary tract infections (UTIs), but recurrent and chronic infections continue to plague many individuals. Most UTIs are caused by strains of uropathogenic Escherichia coli (UPEC), which can form both extra- and intracellular biofilm-like communities within the bladder. UPEC also persist inside host urothelial cells in a more quiescent state, sequestered within late endosomal compartments. Here, we tested a panel of 17 different antibiotics, representing seven distinct functional classes, for their effects on the survival of the reference UPEC isolate UTI89 within both biofilms and host bladder urothelial cells. All but one of the tested antibiotics prevented UTI89 growth in broth culture, and most were at least modestly effective against bacteria present within in vitro-grown biofilms. In contrast, only a few of the antibiotics, including nitrofurantoin and the fluoroquinolones ciprofloxacin and sparfloxacin, were able to eliminate intracellular bacteria in bladder cell culture-based assays. However, in a mouse UTI model system in which these antibiotics reached concentrations in the urine specimens that far exceeded minimal inhibitory doses, UPEC reservoirs in bladder tissues were not effectively eradicated. We conclude that the persistence of UPEC within the bladder, regardless of antibiotic treatments, is likely facilitated by a combination of biofilm formation, entry of UPEC into a quiescent or semiquiescent state within host cells, and the stalwart permeability barrier function associated with the bladder urothelium.

Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.

Aims: adenosine A(2A) and A(2B) receptor subtypes have both been implicated in the modulation of inflammation. We examined adenosine A(2A) and A(2B) receptor expression, signaling pathways and the effect of adenosine A(2A) and A(2B) receptor activation on the uropathogenic Escherichia coli (UPEC)-stimulated IL-8 response in human uroepithelial cells (UROtsa)., Methods: receptor expression was examined by RT-PCR and Western blot, and IL-8 production, intracellular cAMP levels and phosphoproteins were measured by ELISA, EIA and multiplex immunoassay, respectively., Results: the adenosine A(1), A(2A) and A(2B) receptor subtypes were detected in UROtsa cells. The adenosine A(2A) receptor agonist CGS 21680 did not stimulate cAMP production but CREB phosphorylation was slightly increased. The adenosine A(2) receptor agonist CPCA induced a pronounced cAMP and CREB response. Furthermore, CGS 21680 but not CPCA decreased ERK 1/2 and STAT3 phosphorylation. UPEC infection stimulated the host IL-8 production but CPCA or CGS 21680 did not affect UPEC-evoked IL-8 production., Conclusions: our data identified differences in signaling pathways evoked by adenosine A(2A) and A(2B) receptor activation. Activation of the adenosine A(2A) receptor inhibited STAT3 and ERK 1/2 phosphorylation, while the cAMP-CREB pathway was induced by adenosine A(2B) receptor activation. No anti- or proinflammatory effects were found for uroepithelial adenosine A(2A) or A(2B) receptors., (2010 S. Karger AG, Basel.)

Purpose: Bacteriuria is common in patients with neurogenic bladder who are on clean intermittent catheterization for bladder emptying. In a longitudinal study patients carried 1 or 2 clones of Escherichia coli in the urine during months of surveillance. An explanation for persistent bacteriuria in this population could be that periurethral E. coli inoculated during clean intermittent catheterization would attach via type I adhesin, invade superficial bladder epithelial cells and establish reservoirs. Resurgence of bacteria from these reservoirs in bladder epithelium could later reenter the urine and establish a recurrent episode of bacteriuria. We investigated whether bacterial reservoirs were present in the superficial epithelium of patients with neurogenic bladder and chronic bacteriuria., Materials and Methods: Bladder biopsies were obtained from patients with neurogenic bladder and a history of chronic recurrent bacteriuria. Biopsies were fixed in Carnoy's solution to preserve the material overlying the luminal surface of the superficial bladder epithelium. Following fixation biopsies were stained with hematoxylin and eosin to detect intracellular bacterial reservoirs and with periodic acid-Schiff for exopolysaccharide of biofilm. Fluorescence in situ hybridization was done to visualize individual bacteria., Results: No evidence of bacterial reservoirs was found in the superficial bladder epithelium of 9 patients with neurogenic bladder. On hematoxylin and eosin staining epithelium with an intact luminal surface had no intracellular bacterial pods. On periodic acid-Schiff staining no biofilm or collection of exopolysaccharide surrounding bacterial communities was found. No collections or individual bacteria were seen on fluorescence in situ hybridization stained sections examined at 1,000x magnification with oil immersion., Conclusions: Bacterial reservoirs do not appear to be an important source of bacteriuria in patients with chronic recurrent bacteriuria due to neurogenic bladder.

Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.

The transitional epithelium of the bladder normally turns over slowly but upon injury undergoes rapid regeneration fueled by basal uroepithelial stem and/or early progenitor cells (USCs). Little is known about the mechanisms underlying the injury response. We investigate the mechanism of bladder epithelial regeneration in response to infection with uropathogenic E. coli (UPEC). Infection resulted in rapid sloughing of superficial cells, a marked inflammatory response, and a substantial spike in basal cell proliferation. In mice with induced urothelial ablation of a member of the TGF-beta receptor superfamily, bone morphogenetic protein (Bmp)-4 receptor, infection led to aberrant urothelial renewal resulting from a block in USC differentiation into superficial cells. Chemical injury also caused sloughing but no inflammation or USC activation. Together, our study indicates that UPEC infection but not chemical injury activates the USC niche, and Bmp signaling is required for regulation of the USC response to infection.

Aims: The significance of biofilm formation for the clinical picture of urinary tract infections (UTI) is largely unknown. We wanted to find out whether Escherichia coli (E. coli) strains isolated from UTI patients differ in their ability to form biofilms and whether this ability is associated with the clinical presentation of UTI., Material and Methods: 70 E. coli strains were isolated from patients with cystitis (43 strains), pyelonephritis (11 strains) and urosepsis (16 strains) and biofilm formation was assessed on polystyrene microtiter plates by measuring the optical density (OD) of the attached material after 72 h of incubation and crystal violet staining of the bacteria. The formation of organized biofilm structures and the viability of the attached bacteria were verified by scanning electron microscopy and confocal scanning laser microscopy in a subsample of 22 strains., Results: 31% of the E. coli strains formed a biofilm. The strains isolated from patients with pyelonephritis had higher ODs than those from patients with cystitis (difference of the means 0.19, 95% confidence limits (CL) 0.06 - 0.32, p = 0.02). The E. coli strains susceptible to antibiotics had higher ODs than the resistant strains (difference of the means 0.21, 95% CL 0.03 - 0.27, p = 0.016)., Conclusions: The ability of bacteria to persist and grow in a biofilm seems to be one of the important factors in both the resistance to antibiotics and the severity of urinary tract inflammation.

Previous clinical research has suggested that the consumption of cranberry products prevents the adhesion of Escherichia coli to uroepithelial cells by causing changes in bacterial fimbriae. Atomic force microscopy was used to probe the adhesion forces between E. coli (nonfimbriated strain HB101 and the P-fimbriated variant HB101pDC1) and a model surface (silicon nitride), to determine the effect of growth in cranberry products on bacterial adhesion. Bacteria were grown in tryptic soy broth supplemented with either light cranberry juice cocktail (L-CJC) or cranberry proanthocyanidins (PACs). Growth of E. coli HB101pDC1 and HB101 in L-CJC or PACs resulted in a decrease in adhesion forces with increasing number of cultures. In a macroscale bacteria-uroepithelial cell adhesion assay a decrease in bacterial attachment was observed for E. coli HB101pDC1 grown in L-CJC or PACs. This effect was reversible because bacteria that were regrown in cranberry-free medium regained their ability to attach to uroepithelial cells, and their adhesion forces reverted to the values observed in the control condition. Exposure to increasing concentrations of L-CJC resulted in a decrease of bacterial attachment to uroepithelial cells for the P-fimbriated strain after L-CJC treatment (27% by weight) and after PACs treatment (345.8 microg/mL). Cranberry products affect the surface properties, such as fimbriae and lipopolysaccharides, and adhesion of fimbriated and nonfimbriated E. coli. The concentration of cranberry products and the number of cultures the bacteria were exposed to cranberry determines how much the adhesion forces and attachment are altered.

Uropathogenic E. coli (UPEC) expressing type 1 pili underlie most urinary tract infections (UTIs). UPEC adherence to the bladder urothelium induces a rapid apoptosis and exfoliation of terminally differentiated urothelial cells, a critical event in pathogenesis. Of the four major uroplakin proteins that are densely expressed on superficial urothelial cells, UPIa serves as the receptor for type 1-piliated UPEC, but the contributions of uroplakins to cell death are not known. We examined the role of differentiation and uroplakin expression on UPEC-induced cell death. Utilizing in vitro models of urothelial differentiation, we demonstrated induction of tissue-specific differentiation markers including uroplakins. UPEC-induced urothelial cell death was shown to increase with enhanced differentiation but required expression of uroplakin III: infection with an adenovirus encoding uroplakin III significantly increased cell death, while siRNA directed against uroplakin III abolished UPEC-induced cell death. In a murine model of UTI where superficial urothelial cells were selectively eroded to expose less differentiated cells, urothelial apoptosis was reduced, indicating a requirement for differentiation in UPEC-induced apoptosis in vivo. These data suggest that induction of uroplakin III during urothelial differentiation sensitizes cells to UPEC-induced death. Thus, uroplakin III plays a pivotal role in UTI pathogenesis.

Purpose: Catheter associated urinary tract infection is the most common type of hospital acquired infection. An understanding of catheter associated urinary tract infection pathogenesis is needed to improve the control and treatment of these infections. We investigated the relationship among catheter material, bacteria and bladder epithelial cell reaction., Materials and Methods: Urinary catheter sections and a clinical isolate of Escherichia coli were added to human bladder carcinoma epithelial cells in vitro in combination or independently. The catheters were rotated for 30 seconds over the cells, followed by incubation. The cytokines interleukin-6 and 8 were measured by enzyme-linked immunosorbent assay as indicators of inflammation and cell membrane disruption was assessed using a lactate dehydrogenase assay., Results: The levels of lactate dehydrogenase release and cytokine production depended on the number of bacteria added. Bacteria grown for 3 days caused greater secretion of cytokines than bacteria grown overnight. Silicone catheter material alone caused immediate damage to cells with increased lactate dehydrogenase in the supernatant but little interleukin-6 or 8 production. Silicone catheters caused significantly less cytokine secretion from bladder cells than latex catheters. Conversely bacteria caused little immediate damage to cells but stimulated cytokine production after 12 hours., Conclusions: Disruption of bladder epithelial cell membranes in vitro occurred immediately as a result of physical abrasion caused by catheters but delayed inflammation occurred in response to bacterial infection.

In the urinary tract, the innate immune system detects conserved bacterial components and responds to infection by activating the proinflammatory transcription factor NF-kappaB, resulting in cytokine secretion and neutrophil recruitment. Uropathogenic Escherichia coli (UPEC), however, has been shown to evade the host innate immune response by suppressing NF-kappaB activation in urothelial cells, which results in decreased cytokine secretion and increased urothelial apoptosis. To understand the molecular basis of UPEC modulation of inflammation, we performed a genetic screen with UPEC strain NU14 to identify genes which are required for modulation of urothelial cytokine secretion. Disruption of ampG (peptidoglycan permease), waaL (lipopolysaccharide O antigen ligase), or alr (alanine racemase) resulted in increased urothelial interleukin-8 (IL-8) and IL-6 release from urothelial cell cultures. Targeted deletion of these genes also resulted in elevated urothelial cytokine production during UPEC infection. Conditioned media from bacterial cultures of NU14 DeltaampG and NU14 DeltawaaL contained a heat-stable factor(s) which stimulated greater urothelial IL-8 secretion than that in NU14-conditioned medium. In a mouse model of urinary tract infection, NU14 DeltaampG, NU14 DeltawaaL, and NU14 Deltaalr were attenuated compared to wild-type NU14 and showed reduced fitness in competition experiments. Instillation of NU14 DeltaampG or NU14 DeltawaaL increased bladder neutrophil recruitment, indicating that enhanced urothelial cytokine secretion during urinary tract infection results in an altered host response. Thus, UPEC evasion of innate immune detection of bacterial components, such as lipopolysaccharide and peptidoglycan fragments, is likely an important factor in the ability of UPEC to colonize the urinary tract.