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Urinary tract infections (UTIs) are widespread and affect a large portion of the human population. Cranberry juices and extracts have been used for UTI prevention due to their content of bioactive proanthocyanidins (PACs), particularly of the A type (PAC-A). Controversial clinical results obtained with cranberry are often due to a lack of precise determination and authentication of the PAC-A content. This study used Oximacro (Biosfered S.r.l., Turin, Italy), a cranberry extract with a high content of PAC-A, to prevent UTIs in female and male volunteers. The Oximacro PACs content was assayed using the Brunswick Laboratories 4-dimethylaminocinnamaldehyde (BL-DMAC) method, and the dimer and trimer PACs-A and PACs-B percentages were determined via high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). A balanced group of female (ranging from 19 to over 51 years) and male volunteers (over 51 years) was divided into two groups. The experimental group received 1 capsule containing Oximacro (36 mg PACs-A) twice per day (morning and evening) for 7 days, and the placebo group was given the same number of capsules with no PACs. Analysis of Oximacro revealed a high total PAC content (372.34 mg/g ± 2.3) and a high percentage of PAC-A dimers and trimers (86.72% ± 1.65). After 7 days of Oximacro administration, a significant difference was found between the placebo and Oximacro groups for both females (Mann-Whitney U-test = 875; P < .001; n = 60) and males (Mann-Whitney U-test = 24; P = .016; n = 10). When the female and male age ranges were analysed separately, the female age range 31-35 showed only slightly significant differences between the placebo and Oximacro groups (Mann-Whitney U-test = 20.5; P = .095; n = 10), whereas all other female age ranges showed highly significant differences between the placebo and Oximacro groups (Mann-Whitney U-test = 25; P = .008; n = 10). Furthermore, colony forming unit/mL counts from the urine cultures showed a significant difference (P < .001) between the experimental and the placebo groups (SD difference = 51688; df = 34, t = -10.27; Dunn-Sidak Adjusted P < .001, Bonferroni Adjusted P < .001). Careful determination of the total PAC content using the BL-DMAC method and the authentication of PACs-A with mass spectrometry in cranberry extracts are necessary to prepare effective doses for UTI prevention. A dose of 112 mg Oximacro containing 36 mg PACs-A was found to be effective in preventing UTIs when used twice per day for 7 days.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments 1 , 2 . Here we create multilayer bladder ‘assembloids’ by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1–BMP–hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity. Multilayer 3D reconstitution of bladder stem cells with stromal cells enables recapitulation of the architecture and molecular functions of bladder tissue.

In humans and rodent models, commensal gut bacteria contribute to post-stroke infection. Experimental stroke in rodents causes gut barrier dysfunction and permeability, enabling translocation and dissemination of host gut microbiota. Bacterial infection is highly prevalent in patients who have had a stroke. Despite the potential contribution of micro-aspiration in post-stroke pneumonia, we found that the majority of the microorganisms detected in the patients who developed infections after having a stroke were common commensal bacteria that normally reside in the intestinal tracts. In a mouse model of ischemic stroke, post-stroke infection was only observed in mice that were born and raised in specific-pathogen-free facilities; this was not seen in mice that were born and raised in germ-free facilities. Using high-throughput 16S rRNA gene amplicon sequencing and bioinformatics analyses, we provide evidence demonstrating that the source of the bacteria forming the microbial community in the lungs of post-stroke mice was indeed the host small intestine. Additionally, stroke-induced gut barrier permeability and dysfunction preceded the dissemination of orally inoculated bacteria to peripheral tissues. This study identifies a novel pathway in which stroke promotes the translocation and dissemination of selective strains of bacteria that originated from the host gut microbiota.

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 (ΔefaΔmntH1ΔmntH2 strain) led to drastic reductions (>95%) in cellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 (ΔefaΔmntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials.

The pathogenesis of urosepsis in uncomplicated and community-acquired urinary tract infections (UTIs) caused by Escherichia coli was studied. We hypothesized that siderophores involved in iron uptake may determine bacterial adaptation to the urine and blood environment in patients with UTI, leading to urosepsis. E. coli isolates were compared from urosepsis patients (cases) and UTI (control group). The patterns of bacterial DNA fingerprints isolated from the blood and urine of patients with urosepsis were compared by PCR melting profile method to detect urosepsis and exclude nosocomial infection. Chromium Azurol S (CAS) assay and RT-qPCR were used to investigate the expression levels of siderophore genes in artificial urine and M9 supplemented with whole blood. E. coli isolates from artificial urine were proteomically analysed. The pro-inflammatory factors IL-2,4,6,8,10, IFN-gamma, TNF-alpha and CRP were quantified in the patients’ serum. PCR detected and co-occurrence of enterobactin, aerobactin and yersiniabactin was significantly more frequent in the urosepsis (P = 0.0039) and with the iha gene may represent markers of urosepsis risk. Aerobactin was dominant in urosepsis (P = 0.03), but its expression in artificial urine was twice higher than in blood (P = 0.03). When cultured in artificial urine, the expression of entC was significantly higher (P = 0.029), while the expression of iro-2 , iucA and iroB were lower in strains obtained from urosepsis (P = 0.007; P = 0.030; P = 0.012; respectively) as compared to the UTI group. Ferritin-1, iron uptake system component EfeO, ferrous iron transport protein B, nitrate/nitrite response regulator protein NarL, protein HemY, ferrienterobactin receptor FepA, lipopolysaccharide export system protein LptA and 2Fe-2S ferredoxin were found by proteomic analyses in urosepsis only. A positive association of IL-6,8,10, TNF and CRP proteins with urosepsis was observed. In conclusion, risk factors for UTI-related sepsis may be related to the iron uptake system, and genetic and proteomic profiles may help identify them.

Recurrent urinary tract infections (rUTIs) are a source of considerable morbidity in women. The infecting bacteria in both rUTIs and a de novo acute infection have been thought to originate from an extraurinary location. Here, we show in a murine model of UTI that uropathogenic Escherichia coli (UPEC) established quiescent intracellular reservoirs (QIRs) in Lamp1⁺ endosomes within the urinary bladder epithelium. Depending on the integrity of the urothelial barriers at the time of initial infection, these QIRs were established within terminally differentiated superficial facet cells and/or underlying transitional epithelial cells. Treatment of infected bladders harboring exclusively superficial facet cell QIRs with the cationic protein, protamine sulfate, led to epithelial exfoliation and eradication of bacteria in 100% of treated animals. However, when the bacterial QIRs were harbored in underlying transitional cells, stimulation of epithelial turnover triggered reemergence of viable organisms and recurrence of infection. Thus, our results suggest (i) that bacterial QIRs within the bladder may be a previously unappreciated source of recurrent UTIs and (ii) that inducing epithelial exfoliation may be a therapeutic avenue for treating this heretofore recalcitrant disease.

Metabolomics has emerged as a mainstream approach for investigating complex metabolic phenotypes but has yet to be integrated into routine clinical diagnostics. Metabolomics-based diagnosis of urinary tract infections (UTIs) is a logical application of this technology since microbial waste products are concentrated in the bladder and thus could be suitable markers of infection. We conducted an untargeted metabolomics screen of clinical specimens from patients with suspected UTIs and identified two metabolites, agmatine, and N6-methyladenine, that are predictive of culture-positive samples. We developed a 3.2-min LC-MS assay to quantify these metabolites and showed that agmatine and N6-methyladenine correctly identify UTIs caused by 13 Enterobacterales species and 3 non- Enterobacterales species, accounting for over 90% of infections (agmatine AUC > 0.95; N6-methyladenine AUC > 0.89). These markers were robust predictors across two blinded cohorts totaling 1629 patient samples. These findings demonstrate the potential utility of metabolomics in clinical diagnostics for rapidly detecting UTIs. Microbial catabolites in urine provide a rapid method for detecting urinary tract infections (UTIs). Here, the authors describe an LC-MS metabolomics approach for detecting two catabolites collectively produced by 90% of UTI microbes.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) strains are among the most common bacterial infections in humans, causing cystitis, pyelonephritis and, in the absence of appropriate treatment, sepsis. Effective therapies and preventive strategies are still lacking, which highlights the need to better understand UPEC virulence mechanisms. Herein, we describe the role of three groups of bacterial autotransporters (ATs): serine protease autotransporter (SPATE), trimeric autotransporter adhesins (TAA), and autotransporter adhesin AIDA-I, and their possible contribution to the induction of UTI and urosepsis. AT, depending on the type, exhibits functions such as adhesion, serum resistance, hemagglutination, protease activity, biofilm formation and toxin activity. By summarizing the molecular functions of AT proteins, our review highlights their potential as targets for novel therapeutic and preventive approaches against UTIs and urosepsis.

Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts “antibiotic-like” activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH’s capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs.

People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney's innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney's collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell-specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor-mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.