Uropathogenic Escherichia coli infection: innate immune disorder, bladder damage, and Tailin Fang II.

Background: Uropathogenic Escherichia coli (UPEC) activates innate immune response upon invading the urinary tract, whereas UPEC can also enter bladder epithelial cells (BECs) through interactions with fusiform vesicles on cell surfaces and subsequently escape from the vesicles into the cytoplasm to establish intracellular bacterial communities, finally evading the host immune system and leading to recurrent urinary tract infection (RUTI). Tailin Fang II (TLF-II) is a Chinese herbal formulation composed of botanicals that has been clinically proven to be effective in treating urinary tract infection (UTI). However, the underlying therapeutic mechanisms remain poorly understood. Methods: Network pharmacology analysis of TLF-II was conducted. Female Balb/C mice were transurethrally inoculated with UPEC CFT073 strain to establish the UTI mouse model. Levofloxacin was used as a positive control. Mice were randomly divided into four groups: negative control, UTI, TLF-II, and levofloxacin. Histopathological changes in bladder tissues were assessed by evaluating the bladder organ index and performing hematoxylin-eosin staining. The bacterial load in the bladder tissue and urine sample of mice was quantified. Activation of the TLR4-NF-κB pathway was investigated through immunohistochemistry and western blotting. The urinary levels of interleukin (IL)-1ß and IL-6 and urine leukocyte counts were monitored. We also determined the protein expressions of markers associated with fusiform vesicles, Rab27b and Galectin-3, and levels of the phosphate transporter protein SLC20A1. Subsequently, the co-localization of Rab27b and SLC20A1 with CFT073 was examined using confocal fluorescence microscopy. Results: Data of network pharmacology analysis suggested that TLF-II could against UTI through multiple targets and pathways associated with innate immunity and inflammation. Additionally, TLF-II significantly attenuated UPEC-induced bladder injury and reduced the bladder bacterial load. Meanwhile, TLF-II inhibited the expression of TLR4 and NF-κB on BECs and decreased the urine levels of IL-1ß and IL-6 and urine leukocyte counts. TLF-II reduced SLC20A1 and Galectin-3 expressions and increased Rab27b expression. The co-localization of SLC20A1 and Rab27b with CFT073 was significantly reduced in the TLF-II group. Conclusion: Collectively, innate immunity and bacterial escape from fusiform vesicles play important roles in UPEC-induced bladder infections. Our findings suggest that TLF-II combats UPEC-induced bladder infections by effectively mitigating bladder inflammation and preventing bacterial escape from fusiform vesicles into the cytoplasm. The findings suggest that TLF-II is a promising option for treating UTI and reducing its recurrence.

Study on the therapeutic mechanism of HJ granules in a rat model of urinary tract infection caused by Escherichia coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Urinary tract infections (UTIs) are globally prevalent infectious diseases, predominantly caused by uropathogenic Escherichia coli (UPEC). The misuse of antibiotics has led to the emergence of several drug-resistant strains. Traditional Chinese Medicine (TCM) has its own advantages in the treatment of UTIs. HJ granules is a herbal formula used for the treatment of UTIs. However, its mechanism of action is not clear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic efficacy and mechanism of action of HJ granules in a rat model of UTI caused by Escherichia coli (E coli) CFT073. MATERIALS AND METHODS: SD rats were selected to establish a rat UTI model by injecting UPEC strain CFT073 into the bladder using the transurethral placement method. HJ granules were administered to rats after modelling and the efficacy of HJ granule was investigated by measuring urinary decanalogue, inflammatory factors in bladder tissue and pathological changes in the bladder after 3d of administration. Expression of sonic hedgehog (SHH), NOD-like receptor thermoprotein domain 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and activation of cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by western blotting and immunofluorescence staining in rat bladder tissue. NLRP3, ASC and caspase-1, a cysteine-containing aspartic protein, were expressed and activated. RESULTS: The results showed that infection of rats with UPEC resulted in increased pH and erythrocytes in bladder irrigation fluid; increased expression of IL-1ß, IL-6 and SHH and decreased expression of IL-10 in bladder tissue; and significant upregulation of the expression of both SHH and NLRP3 inflammasom and significant activation of NLRP3 inflammasom. HJ granules significantly increased the concentration of IL-10 in the bladder, inhibited the expression of SHH and NLRP3 inflammasom in bladder tissue, and suppressed the activation of NLRP3 inflammasom, thereby reducing inflammatory lesions in bladder tissue. CONCLUSION: HJ granules may improve bladder injury and treat UTIs by inhibiting the expression and activation of NLRP3 inflammasom.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Differential expression of urease genes and ureolytic activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa isolates in different nutritional conditions.

Uropathogens have adaptation strategies to survive in the host urinary tract by efficiently utilizing and tolerating the urinary metabolites. Many uropathogens harbour the enzyme urease for the breakdown of urea and the enzymatic breakdown of urea increases the pH and facilitate the struvite crystallization. In this study, the differential urease activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa strains was investigated under different nutritional conditions. The experiments included measurement of growth, pH, urease activity, NH4-N generation and urease gene (ureC) expression among the bacterial strains under different conditions. Further, the implications of urea breakdown on the struvite crystallization in vitro and biofilm formation were also assessed. The study included urease positive isolates and for comparison urease negative isolates were included. Compared to the urease negative strains the urease positive strains formed higher biofilms and motility. The urease positive P. aeruginosa showed significantly higher (p < 0.01) pH and urease activity (A557-A630) compared to E. coli under experimental conditions. Further, supplementation of glucose to the growth media significantly increased the urease activity in P. aeruginosa and in contrast, it was significantly lower in E. coli. The expression profile of urease gene (ureC) was significantly higher (p < 0.001) in P. aeruginosa compared to E. coli and was consistent with the biochemical results of the urease activity under the nutritional conditions. The differential urease activity under two nutritional conditions influenced the biogenic struvite crystallization. It correlated with the urease activity showing higher crystallization rate in P. aeruginosa compared to E. coli. The results highlight the differential urease activity in two common uropathogens under different nutritional conditions that may have significant role on the regulation of virulence, pathogenicity and in the kidney stone disease.

Urinary Tract Infections Caused by Uropathogenic Escherichia coli: Mechanisms of Infection and Treatment Options.

Urinary tract infections (UTIs) are common bacterial infections that represent a severe public health problem. They are often caused by Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumonia), Proteus mirabilis (P. mirabilis), Enterococcus faecalis (E. faecalis), and Staphylococcus saprophyticus (S. saprophyticus). Among these, uropathogenic E. coli (UPEC) are the most common causative agent in both uncomplicated and complicated UTIs. The adaptive evolution of UPEC has been observed in several ways, including changes in colonization, attachment, invasion, and intracellular replication to invade the urothelium and survive intracellularly. While antibiotic therapy has historically been very successful in controlling UTIs, high recurrence rates and increasing antimicrobial resistance among uropathogens threaten to greatly reduce the efficacy of these treatments. Furthermore, the gradual global emergence of multidrug-resistant UPEC has highlighted the need to further explore its pathogenesis and seek alternative therapeutic and preventative strategies. Therefore, a thorough understanding of the clinical status and pathogenesis of UTIs and the advantages and disadvantages of antibiotics as a conventional treatment option could spark a surge in the search for alternative treatment options, especially vaccines and medicinal plants. Such options targeting multiple pathogenic mechanisms of UPEC are expected to be a focus of UTI management in the future to help combat antibiotic resistance.

Ferritinophagy-mediated iron competition in RUTIs: Tug-of-war between UPEC and host.

Uropathogenic Escherichia coli (UPEC) is the main pathogen of recurrent urinary tract infections (RUTIs). Urinary tract infection is a complicated interaction between UPEC and the host. During infection, UPEC can evade the host's immune response and retain in bladder epithelial cells, which requires adequate nutritional support. Iron is the first necessary trace element in life and a key nutritional factor, making it an important part of the competition between UPEC and the host. On the one hand, UPEC grabs iron to satisfy its reproduction, on the other hand, the host relies on iron to build nutritional immunity defenses against UPEC. Ferritinophagy is a selective autophagy of ferritin mediated by nuclear receptor coactivator 4, which is not only a way for the host to regulate iron metabolism to maintain iron homeostasis, but also a key point of competition between the host and UPEC. Although recent studies have confirmed the role of ferritinophagy in the progression of many diseases, the mechanism of potential interactions between ferritinophagy in UPEC and the host is poorly understood. In this paper, we reviewed the potential mechanisms of ferritinophagy-mediated iron competition in the UPEC-host interactions. This competitive relationship, like a tug-of-war, is a confrontation between the capability of UPEC to capture iron and the host's nutritional immunity defense, which could be the trigger for RUTIs. Therefore, understanding ferritinophagy-mediated iron competition may provide new strategies for exploring effective antibiotic alternative therapies to prevent and treat RUTIs.

Uropathogenic Escherichia coli endeavors: an insight into the characteristic features, resistance mechanism, and treatment choice.

Uropathogenic Escherichia coli (UPEC) are the strains diverted from the intestinal status and account mainly for uropathogenicity. This pathotype has gained specifications in structure and virulence to turn into a competent uropathogenic organism. Biofilm formation and antibiotic resistance play an important role in the organism's persistence in the urinary tract. Increased consumption of carbapenem prescribed for multidrug-resistant (MDR) and Extended-spectrum-beta lactamase (ESBL)-producing UPECs, has added to the expansion of resistance. The World Health Organization (WHO) and Centre for Disease Control (CDC) placed the Carbapenem-resistant Enterobacteriaceae (CRE) on their treatment priority lists. Understanding both patterns of pathogenicity, and multiple drug resistance may provide guidance for the rational use of anti-bacterial agents in the clinic. Developing an effective vaccine, adherence-inhibiting compounds, cranberry juice, and probiotics are non-antibiotical approaches proposed for the treatment of drug-resistant UTIs. We aimed to review the distinguishing characteristics, current therapeutic options and promising non-antibiotical approaches against ESBL-producing and CRE UPECs.

Seven-day Oral Intake of Orthosiphon stamineus Leaves Infusion Exerts Antiadhesive Ex Vivo Activity Against Uropathogenic E. coli in Urine Samples.

Orthosiphon stamineus leaves (Java tea) extract is traditionally used for the treatment of urinary tract infections. According to recent in vitro data, animal infection studies, and transcriptomic investigations, polymethoxylated flavones from Java tea exert antiadhesive activity against uropathogenic Escherichia coli (UPEC). This antiadhesive activity has been shown to reduce bladder and kidney lesion in a mice infection model. As no data on the antivirulent activity of Java tea intake on humans are available, a biomedical study was performed on 20 healthy volunteers who self-administered Orthosiphon infusion (4 × 3 g per day, orally) for 7 days. The herbal material used for the study conformed to the specification of the European Pharmacopoeia, and ultra high-performance liquid chromatography (UHPLC) of the infusion showed rosmarinic acid, caffeic acid, and cichoric acid to be the main compounds aside from polymethoxylated flavones. Rosmarinic acid was quantified in the tea preparations with 243 ± 22 µg/mL, indicating sufficient reproducibility of the preparation of the infusion. Urine samples were obtained during the biomedical study on day 1 (control urine, prior to Java tea intake), 3, 6 and 8. Antiadhesive activity of the urine samples was quantified by flowcytometric assay using pre-treated UPEC NU14 and human T24 bladder cells. Pooled urine samples indicated significant inhibition of bacterial adhesion on day 3, 6 and 8. The urine samples had no influence on the invasion of UPEC into host cells. Bacterial proliferation was slightly reduced after 24 h incubation with the urine samples. Gene expression analysis (qPCR) revealed strong induction of fitness and motility gene fliC and downregulation of hemin uptake system chuT. These data correlate with previously reported datasets from in vitro transcriptomic analysis. Increased bacterial motility was monitored using a motility assay in soft agar with UPEC UTI89. The intake of Java tea had no effect on the concentration of Tamm-Horsfall Protein in the urine samples. The present study explains the antiadhesive and anti-infective effect of the plant extract by triggering UPEC from a sessile lifestyle into a motile bacterial form, with reduced adhesive capacity.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44 kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29 cells with the chimeric protein was significantly higher than the stimulated Ht29 cells with CNF-1 alone and un-stimulated Ht29 cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Polysaccharides from Vaccaria segetalis seeds reduce urinary tract infections by inhibiting the adhesion and invasion abilities of uropathogenic Escherichia coli.

The seeds of Vaccaria segetalis (Neck.) are from a traditional medicinal plant Garcke, also called Wang-Bu-Liu-Xing in China. According to the Chinese Pharmacopoeia, the seeds of V. segetalis can be used for treating urinary system diseases. This study was designed to investigate the underlying mechanism of VSP (polysaccharides from Vaccaria segetalis) against urinary tract infections caused by uropathogenic Escherichia coli (UPEC). Here, both in vitro and in vivo infection models were established with the UPEC strain CFT073. Bacterial adhesion and invasion into bladder epithelial cells were analyzed. We found that VSP reduced the adhesion of UPEC to the host by inhibiting the expression of bacterial hair follicle adhesion genes. VSP also reduced the invasion of UPEC by regulating the uroplakins and Toll-like receptors of host epithelial cells. In addition, the swarming motility and flagella-mediated motility genes flhC, flhD and Flic of UPEC were diminished after VSP intervention. Taken together, our findings reveal a possible mechanism by which VSP interferes with the adhesion and invasion of UPEC.

UPEC Colonic-Virulence and Urovirulence Are Blunted by Proanthocyanidins-Rich Cranberry Extract Microbial Metabolites in a Gut Model and a 3D Tissue-Engineered Urothelium.

Uropathogenic Escherichia coli (UPEC) ecology-pathophysiology from the gut reservoir to its urothelium infection site is poorly understood, resulting in equivocal benefits in the use of cranberry as prophylaxis against urinary tract infections. To add further understanding from the previous findings on PAC antiadhesive properties against UPEC, we assessed in this study the effects of proanthocyanidins (PAC) rich cranberry extract microbial metabolites on UTI89 virulence and fitness in contrasting ecological UPEC's environments. For this purpose, we developed an original model combining a colonic fermentation system (SHIME) with a dialysis cassette device enclosing UPEC and a 3D tissue-engineered urothelium. Two healthy fecal donors inoculated the colons. Dialysis cassettes containing 7log10 CFU/mL UTI89 were immersed for 2h in the SHIME colons to assess the effect of untreated (7-day control diet)/treated (14-day PAC-rich extract) metabolomes on UPEC behavior. Engineered urothelium were then infected with dialysates containing UPEC for 6 h. This work demonstrated for the first time that in the control fecal microbiota condition without added PAC, the UPEC virulence genes were activated upstream the infection site, in the gut. However, PAC microbial-derived cranberry metabolites displayed a remarkable propensity to blunt activation of genes encoding toxin, adhesin/invasins in the gut and on the urothelium, in a donor-dependent manner. Variability in subjects' gut microbiota and ensuing contrasting cranberry PAC metabolism affects UPEC virulence and should be taken into consideration when designing cranberry efficacy clinical trials. IMPORTANCE Uropathogenic Escherichia coli (UPEC) are the primary cause of recurrent urinary tract infections (UTI). The poor understanding of UPEC ecology-pathophysiology from its reservoir-the gut, to its infection site-the urothelium, partly explains the inadequate and abusive use of antibiotics to treat UTI, which leads to a dramatic upsurge in antibiotic-resistance cases. In this context, we evaluated the effect of a cranberry proanthocyanidins (PAC)-rich extract on the UPEC survival and virulence in a bipartite model of a gut microbial environment and a 3D urothelium model. We demonstrated that PAC-rich cranberry extract microbial metabolites significantly blunt activation of UPEC virulence genes at an early stage in the gut reservoir. We also showed that altered virulence in the gut affects infectivity on the urothelium in a microbiota-dependent manner. Among the possible mechanisms, we surmise that specific microbial PAC metabolites may attenuate UPEC virulence, thereby explaining the preventative, yet contentious properties of cranberry against UTI.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Aqueous extract from Equisetum arvense stimulates the secretion of Tamm-Horsfall protein in human urine after oral intake.

BACKGROUND: Within European traditional phytotherapy, extracts from different herbal plants are used for prevention and therapy of uncomplicated urinary tract infections and for flushing out of kidney grits. Besides increased urine flow by slight diuretic effects, also stimulation of Tamm-Horsfall protein (syn. THP, uromodulin) in the distal part of the kidney could explain reduced kidney gravel and anti-virulent activity against uropathogenic E. coli. PURPOSES: Evaluation of THP-inducing activity of extracts from Equisetum arvense, Levisticum officinalis, Ilex paraguariensis, Juniperus communis, Urtica dioica, and Taraxacum officinale by quantification of THP in urine samples after oral application to humans. STUDY DESIGN: 7 days p.o. application of the test intervention to healthy volunteers (n = 10 per intervention group) and analysis of urine samples at day 1 (untreated control values), and days 3, 6 and 8 on THP content by validated ELISA. Antiadhesive activity of urine samples was monitored by flow cytometry using UPEC strain NU14 against human T24 bladder cells. RESULTS: An aqueous extract from E. arvense, fully characterized by a specific LC-MS method, induced THP concentration in urine samples significantly during a 7-day p.o. application up to 300%, related to the untreated controls. Ex vivo investigation of the individual and pooled urine samples with elevated THP concentrations showed good correlation to antiadhesive effects against UPEC NU14 to T24 cells. Urine samples of the Equisetum treated volunteers had no effect on the proliferation and on biofilm formation of UPEC NU14. Silica excretion in the urine samples had no correlation to the respective THP levels. Monitoring of electrolyte content in the urine samples indicat ed diuretic effects of the intervention with Equisetum extract. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 80 compounds from the extract by MS evaluations and 18 compounds by UV detection. This protocol will provide a valuable tool for future quality control of Equisetum extract. CONCLUSION: Aqueous extract from E. arvense significantly stimulates THP secretion in urine samples after 7 days of oral intake and inhibits the interplay between UPEC and bladder host cells. This could explain the therapeutic use of this herbal material for urinary tract infections and kidney gravel. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 82% of all eluted compounds. This protocol will provide a valuable tool for future quality control of Equisetum extract.

Glycosuria Alters Uropathogenic Escherichia coli Global Gene Expression and Virulence.

Uropathogenic Escherichia coli (UPEC) is the principal etiology of more than half of urinary tract infections (UTI) in humans with diabetes mellitus. Epidemiological data and studies in mouse model of ascending UTI have elucidated various host factors responsible for increasing the susceptibility of diabetic hosts to UPEC-UTI. In contrast, diabetic urinary microenvironment-mediated alterations in UPEC physiology and its contributions to shaping UPEC-UTI pathogenesis in diabetes have not been examined. To address our central hypothesis that glycosuria directly induces urinary virulence of UPEC, we compared virulence characteristics and gene expression in human UPEC strains UTI89 (cystitis) and CFT073 (pyelonephritis), exposed for 2 h in vitro to urine from either male or female donors that was either plain or supplemented with glucose to mimic glycosuria. Compared to control UPEC exposed to nutrient-rich culture medium, lysogeny broth, glycosuria-exposed UPEC exhibited significant increase in biofilm formation and reduction in the hemagglutination of Guinea pig erythrocytes (a measure of type 1 piliation). In addition, the analysis of UTI89 transcriptome by RNA sequencing revealed that 2-h-long, in vitro exposure to glycosuria also significantly alters expression of virulence and metabolic genes central to urinary virulence of UPEC. Addition of galactose as an alternative carbon source affected biofilm formation and gene expression profile of UPEC to an extent similar to that observed with glucose exposure. In summary, our results provide novel insights into how glycosuria-mediated rapid changes in UPEC fitness may facilitate UTI pathogenesis in the diabetic urinary microenvironment. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is an important causative agent of urinary tract infections in diabetic humans. We examined the effects of in vitro exposure to glycosuria (presence of glucose in urine) on the virulence and gene expression by UPEC. Our results show that glycosuria rapidly (in 2 h) alters UPEC gene expression, induces biofilm formation, and suppresses type 1 piliation. These results offer novel insights into the pathogenesis of UPEC in the urinary tract.

Studies on Virulence and Extended-Spectrum ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates and Therapeutic Effect of Fosfomycin in Acute Pyelonephritis Mice.

The understanding about virulence factors (VFs) and the drug resistance of uropathogenic Escherichia coli (UPEC) helps us understand the pathogenesis of urinary tract infections (UTIs) and make better decisions for clinical treatment. This study examined the correlation between the extended-spectrum ß-lactamases (ESBLs) phenotype and VFs in UPEC strains. In addition, we validated the therapeutic potential of fosfomycin in acute pyelonephritis mice. From May 2017 to November 2018, 22 nonduplicate E coli. strains were isolated from UTI patients. PCR was utilized to detect the distribution of virulence genes. We also analyzed the ESBL phenotype in E coli. We further evaluated the therapeutic effect of intravenous fosfomycin treatment in the acute pyelonephritis (APN) model. All 22 UPEC strains expressed the type 1 fimbriae (FimH) gene and more than 50% (12/22) of strains produced ESBLs. The detection rates of the iron acquisition-associated genes ChuT and IutA were 77.3% (n = 17) and 50% (n = 11) and those of P fimbria papA and papC genes were 45% (n = 10) and 50% (n = 11), respectively. Though the VFs were closely related with pathologenicity, the relationship between VFs and ESBLs still needs further investigation. Furthermore, intravenous fosfomycin 800 mg/kg significantly reduced the bacterial load and the inflammatory infiltration in the bladder and kidney, maintaining the structural integrity of the kidney. Intravenous fosfomycin administration can be used for the treatment of acute pyelonephritis caused by highly pathogenic and drug-resistant UPEC strains.

Dictamnine Inhibits the Adhesion to and Invasion of Uropathogenic Escherichia Coli (UPEC) to Urothelial Cells.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

Characterization of PACs profile and bioactivity of a novel nutraceutical combining cranberry extracts with different PAC-A oligomers, D-mannose and ascorbic acid: An in vivo/ex vivo evaluation of dual mechanism of action on intestinal barrier and urinary epithelium.

In this paper, an A-type procyanidin (PAC)-rich cranberry extract (CB-B) was obtained mixing different extracts and was formulated with D-mannose and ascorbic acid to obtain a novel nutraceutical (URO-F) aimed at preventing non-complicated bacterial urinary tract infections (UTIs). To assess the bioactivity of CB-B and URO-F, urine samples collected from six healthy volunteers undergoing a 2-days oral consumption of 0.41 g/day of CB-B or 10 g/day of URO-F (corresponding to 72 mg/day of PACs) were tested against uropathogenic E. coli (UPEC) incubated on urinary bladder epithelial cells (T24). Urinary markers of CB-B and URO-F consumption were assessed in the same urine output by UPLC-QTOF-based untargeted metabolomics approach. CB-B and URO-F were evaluated for their ability to promote the intestinal barrier function by restoring the trans-epithelial electrical resistance (TEER) and to inhibit the production of inflammatory cytokines in intestinal epithelial Caco2 cells. CB-B was characterized by a high PAC-A content (70% of total PACs) and a broad distribution of different PACs polymers (dimers-hexamers). Urine from subjects consuming CB-B and URO-F showed a significant effect in reducing the adhesion of UPEC to urothelium in vitro, supporting their efficacy as anti-adhesive agents after oral intake. CB-B inhibited the release of cytokine IL-8, and both products were effective in restoring the TEER. Overall, our results show that the beneficial effects of CB-B and URO-F on UTIs are not only due to the antiadhesive activity of cranberry on UPEC in the urothelium, but also to a multi-target activity involving anti-inflammatory and permeability-enhancing effects on intestinal epithelium.

Diverse anti-inflammation and anti-cancer polyketides isolated from the endophytic fungi Alternaria sp. MG1.

Six new polyketides, alternaritins A-D [(±)-1-4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1-9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

Phytochemical analysis of medicinal plants of Nepal and their antibacterial and antibiofilm activities against uropathogenic Escherichia coli.

BACKGROUND: A biofilm is an extracellular polymeric substance (EPS) composed of polysaccharides, proteins, nucleic acids, and lipids that impede antibiotics and immune cells, thus providing a shielded environment for bacterial growth. Due to biofilm formation, some microbes can show up to 1000 fold increased resistance towards the antibiotics than the normal planktonic forms. The study was conducted to screen the crude extracts of medicinal plants used in Nepal for their in vitro antibiofilm activities. METHODS: Total phenolic and total flavonoid contents were determined by using a Folin-Ciocalteau reagent and aluminium trichloride method, respectively. Resazurin assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The initial antibiofilm activities and their inhibitory concentration (IC50) values were determined by the microtiter based modified crystal violet staining method. RESULTS: Out of 25 different plant extracts were used for the study, methanolic extracts of 20 plants showed a biofilm inhibition activity against five different strong biofilm producing Escherichia coli strains. Calotropis gigantea exhibited inhibition against all five different E. coli strains with IC50 values ranging from 299.7 ± 20.5 to 427.4 ± 2.7 µg/mL. Apart from that, Eclipta prostrata also showed biofilm formation inhibition, followed by Eupatorium adenophorum, Moringa oleifera, Ocimum tenuifolium, Oxalis lantifolia, Prunus persica, and Urtica parviflora. The extracts of C. gigantea, E. prostrata, Mangifera indica, O. tenuifolium, P. persica, and U. parviflora exhibited a moderate to poor MIC value ranging from 625 to 2500 µg/mL. The highest amount of phenolic content (TPC) was found in Acacia catechu followed by Morus alba, which was 38.9 and 25.1 mg gallic acid equivalents, respectively. The highest amount of flavonoid content was found in A. catechu followed by M. indica, which was 27.1 and 20.8 mg quercetin equivalents, respectively. CONCLUSION: Extracts of C. gigantea, E. prostrata, P. persica, U. parviflora, and O. tenuifolium showed antibacterial as well as antibiofilm activity against pathogenic and strong biofilm producing E. coli. Thus, extracts or the pure compound from these medicinal plants could be used as antibiotics in the future.

Immunization with recombinant protein Ag43::UpaH with alum and 1,25(OH)2D3 adjuvants significantly protects Balb/C mice against urinary tract infection caused by uropathogenic Escherichia coli.

The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Designing a vaccine will certainly reduce the occurrence of infection and antibiotic resistance of the isolates. Antigen 43 (Ag43) and autotransporter H (UpaH) have been associated with the virulence of UPEC. In the present study, the efficacy of different formulations of a hybrid protein composed of Ag43 and UpaH with and without alum and 1,25(OH)2D3 (Vitamin D3) adjuvants were evaluated in mice model. A significant increase in IgG and cellular responses was developed against Ag43::UpaH as compared to the control mice. The addition of alum or a mixture of alum and Vitamin D3 to the protein significantly enhanced the serum IgG responses and tended to remain in a steady state until 6 months. In addition, the mentioned formulations produced significant amounts of IgG1, IL-4, and IL-17 as compared to the fusion protein alone. In addition to the mentioned formulations, the combination of protein with Vitamin D3 also resulted in significantly higher serum IgA and IFN-γ levels as compared to the fusion protein alone. Mice immunized with fusion plus alum and formulation protein admixed with both alum and Vitamin D3 significantly reduced the bacterial load in the bladders and kidneys of mice as compared to the control. In this study, for the first time, the ability of a novel hybrid protein in combination with adjuvants alum and Vitamin D3 was evaluated against UPEC. Our results indicated that fusion Ag43::UpaH admixed with alum and Vitamin D3 could be a promising candidate against UTIs.