Urinary Tract Infection Model Research Articles

Repurposing of FDA approved drugs against uropathogenic Escherichia coli: In silico, in vitro, and in vivo analysis

Urinary tract infections (UTIs) are a serious health concern worldwide. Treatment of UTIs is becoming a challenge as uropathogenic Escherichia coli (UPEC), which is the most common etiological agent, has developed resistance to the main classes of antibiotics. Small molecules that interfere with metabolic processes rather than growth are attractive alternatives to conventional antibiotics. Repurposing of already known drugs for treating infectious diseases could be an attractive avenue for finding novel therapeutics against infections caused by UPEC. Virtual screenings enable the rapid and economical identification of target ligands from large libraries of compounds, reducing the cost and time of traditional drug discovery. Moreover, the drugs that have been approved by the FDA have low cytotoxicity and good pharmacological characteristics. In this work, we targeted the HisC enzyme of the histidine biosynthetic pathway as enzymes of this pathway are absent in humans. We screened the library of FDA-approved drugs against HisC via molecular docking, and four hits (Docetaxel, Suramin, Digitoxin, and Nystatin) showing the highest binding energy were selected. These were further tested for antibacterial activity, which was observed only for Docetaxel (MIC value of 640 μg/ml); therefore, Docetaxel was further tested for its efficacy in vivo in murine catheter UTI model and antibiofilm activity using crystal violet staining and scanning electron microscopy. Docetaxel inhibited biofilm formation and reduced the bacterial load in urine, kidney, and bladder. Docking studies revealed that Docetaxel acts by blocking the binding site of HisC to the native substrate by competitive inhibition. Docetaxel may be a potential new inhibitor for UPEC with antibacterial and antibiofilm capability.

Prediction Model for Urinary Tract Infection in Pediatric Urological Surgery Patients

BackgroundUrinary tract infection (UTI) is a common complication in pediatric urological surgery patients and is associated with long-term sequelae, including subsequent recurrent infections and renal scarring. In this study, we aimed to explore the risk factors for UTI in pediatric urological surgery patients and construct a predictive model for UTI.Materials and MethodsA total of 2,235 pediatric patients who underwent urological surgery at a tertiary hospital between February 2019 and January 2020 were included. A multivariate logistic regression model was applied to identify the predictive factors, and a predictive model was constructed using a receiver operating characteristic curve. A multifactorial predictive model was used to categorize the risk of UTI based on the weight of the evidence.ResultsA total of 341 patients with UTI were identified, which corresponded to a prevalence of 15.26% in pediatric urological surgery patients. Multivariate analysis identified six significant risk factors for UTI, including age <12.0 months, upper urinary tract disease, not using an indwelling drainage tube, hospital stay ≥10 days, administration of two or more types of antibiotics, and stent implantation. A combination of the aforementioned factors produced an area under the curve value of 88.37% for preventing UTI in pediatric urological surgery patients. A multifactorial predictive model was created based on the combination of these factors.ConclusionsThe constructed multifactorial model could predict UTI risk in pediatric urological surgery patients with a relatively high predictive value.

Early warning model construction and validation for urinary tract infection in patients with neurogenic lower urinary tract dysfunction (NLUTD): a retrospective study.

BackgroundThis study was performed to construct and validate an early risk warning model of urinary tract infection in patients with neurogenic lower urinary tract dysfunction (NLUTD).MethodsEligible patients with NLUTD admitted to Shenzhen Longcheng hospital from January 2017 to June 2021 were recruited for model construction, internal validation and external validation. The first time point of data collection was within half a month of patients first diagnosed with NLUTD. The second time point was at the 6-month follow-up. The early warning model was constructed by logistic regression. The model prediction effects were validated using the area under the Receiver Operating Characteristic curve, the Boostrap experiment and the calibration plot of the combined data. The model was externally validated using sensitivity, specificity and accuracy.ResultsSix predictors were identified in the model, namely patients ≥65 years old (OR = 2.478, 95%CI [1.215– 5.050]), female (OR = 2.552, 95%CI [1.286–5.065]), diabetes (OR = 2.364, 95%CI) [1.182–4.731]), combined with urinary calculi (OR = 2.948, 95%CI [1.387–6.265]), indwelling catheterization (OR = 1.988, 95%CI [1.003 –3.940]) and bladder behavior training intervention time ≥2 weeks (OR = 2.489, 95%CI [1.233–5.022]); and the early warning model formula was Y = 0.907 × age+ 0.937 × sex + 0.860 × diabetes +1.081 × combined with urinary calculi+ 0.687 × indwelling catheterization+ 0.912 × bladder behavior training intervention time-2.570. The results show that the area under the ROC curve is 0.832, which is close to that of 1,000 Bootstrap internal validation (0.828). The calibration plot shows that the early warning model has good discrimination ability and consistency. The external validation shows the sensitivity is 62.5%, the specificity is 100%, and the accuracy is 90%.ConclusionThe early warning model for urinary tract infection in patients with NLUTD is suitable for clinical practice, which can provide targeted guidance for the evaluation of urinary tract infection in patients with NLUTD.

Screening Biomarkers and Constructing a Predictive Model for Symptomatic Urinary Tract Infection and Asymptomatic Bacteriuria in Patients Undergoing Cutaneous Ureterostomy: A Metagenomic Next-Generation Sequencing Study.

Objectives To investigate the clinical diagnostic value of differential flora as biomarkers in patients with symptomatic urinary tract infection (UTI) and asymptomatic bacteriuria (ASB) undergoing cutaneous ureterostomy based on metagenomic next-generation sequencing and construct predictive models to provide a scientific reference for clinical diagnosis and treatment. Material and Methods. According to standard procedures, samples were taken from each patient for routine tests (urine, ureteral stent, and skin swab around the stoma). Cytokine levels in the blood were also detected. Urinary microflora were measured by mNGS, and potential biomarkers for distinguishing UTI and ASB were identified by differential flora. Finally, we generated the predictive models for ASB and UTI using the Lasso method and cytokine levels. Results Urine culture was performed for 50 patients with cutaneous ureterostomy; 44 of these patients developed bacteriuria. The incidence of symptomatic bacteriuria was 54.55%. Biomarker analysis showed that Propionimicrobium lymphophilum, Staphylococcus haemolyticus, Stenotrophomonas maltophilia, Ralstonia insidiosa, and Aspergillus sydowii all had good predictive performance and were combined in a single model. The predictive model exhibited good prediction performance (area under the curve (AUC) = 0.8729, sensitivity = 80%, specificity = 83.3%, and cutoff = 1.855). We also identified a significant negative correlation between the weight sum of the abundance for these five characteristic pathogens (Sum_weighted_Reads) and levels of the cytokine IL-6 and IL-1β (P < 0.05). Conclusion mNGS had a higher positive detection rate for pathogens in urine samples. The selected differential bacteria can be used as biomarkers of ASB and UTI, and the prediction model has good predictive performance. Analysis also showed that the occurrence of symptoms was related to individual immunity. Combined with the Sum_weighted_Reads cutoff and cytokine levels (IL-6 and IL-1β) of differential flora, it was possible to judge the severity of symptoms in cutaneous ureterostomy patients with bacteriuria and provide new insights for the treatment and intervention of ASB and UTI.

Uropathogenic Escherichia coli can cause cystitis at extremely low inocula in a pig model.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. Experimental models that accurately reflect the high susceptibility to UTI in humans have, however, been lacking. This situation has limited detailed research into the early bladder colonization by uropathogens and the early innate defence mechanisms elicited to prevent this. We recently presented a model of urinary tract infection in pigs, animals that are naturally susceptible to UTI and have greater similarity to the physiology and anatomy of the human urinary tract than traditional rodent UTI models. In the current study, we used the pig model to investigate the minimal infectious inoculum of uropathogenic Escherichia coli, the most common cause of urinary tract infection. We show that in this animal a few individual bacteria that come into contact with the urothelium can give rise to fulminant cystitis, indicating the high infectious potential of uropathogenic E. coli.

Oral fosfomycin activity against Klebsiella pneumoniae in a dynamic bladder infection in vitro model.

IntroductionThe use of oral fosfomycin for urinary tract infections (UTIs) caused by non-Escherichia coli uropathogens is uncertain, including Klebsiella pneumoniae, the second most common uropathogen.MethodsA multicompartment bladder infection in vitro model was used with standard media and synthetic human urine (SHU) to simulate urinary fosfomycin exposure after a single 3 g oral dose (fAUC0–72 16884 mg·h/L, t½ 5.5 h) against 15 K. pneumoniae isolates including ATCC 13883 (MIC 2 to >1024 mg/L) with a constant media inflow (20 mL/h) and 4-hourly voiding of each bladder. The impact of the media (CAMHB + G6P versus SHU) on fosfomycin MIC measurements, drug-free growth kinetics and regrowth after fosfomycin administration was assessed. A low and high starting inoculum (5.5 versus 7.5 log10 cfu/mL) was assessed in the bladder infection model.ResultsCompared with CAMHB, isolates in SHU had a slower growth rate doubling time (37.7 versus 24.1 min) and reduced growth capacity (9.0 ± 0.3 versus 9.4 ± 0.3 log10 cfu/mL), which was further restricted with increased inflow rate (40 mL/h) and more frequent voids (2-hourly). Regrowth was commonly observed in both media with emergence of fosfomycin resistance promoted by a high starting inoculum in CAMHB (MIC rise to ≥1024 mg/L in 13/14 isolates). Resistance was rarely detected in SHU, even with a high starting inoculum (MIC rise to ≥1024 mg/L in 2/14 isolates).ConclusionsSimulated in an in vitro UTI model, the regrowth of K. pneumoniae urinary isolates was inadequately suppressed following oral fosfomycin therapy. Efficacy was further reduced by a high starting inoculum.

External validation and updating of a model to predict urinary tract infection after urogynecologic surgery

To externally validate and update a previously published model predicting postoperative urinary tract infection (UTI) after benign hysterectomy in a geographically distinct cohort of women undergoing urogynecologic surgery. The validation cohort included 351 women who underwent urogynecologic surgery at an academic health system between January and August 2019. Individuals who underwent sacral neuromodulation, non-pelvic floor surgeries, and repeat surgery within 6 months were excluded. Clinical and surgical characteristics were abstracted from the medical record. Primary outcome was incidence of UTI up to 6 weeks after surgery, defined as: UTI symptoms with positive urine culture, physician diagnosis, or decision to treat with antibiotics per the National Surgical Quality Improvement Program guidelines. Missing predictor values were imputed. Predicted probabilities of postoperative UTI were generated for the validation cohort using the original model described by El-Nashar et al. This model included 4 predictors: cystocele, menopausal status, hormone therapy (HT), and preoperative postvoid residual volume >150mL. Predicted probabilities were compared to actual events to determine model performance. Accuracy was measured using the concordance index (C-index), calibration curves, and the Brier score. Model coefficients and intercepts were subsequently refit to an updated model that included additional clinically relevant candidate predictors which were selected using redundancy analysis and variable clustering. Internal validation of the updated model was performed using bootstrapping to correct for bias. Decision curves were used to identify the range of threshold probabilities in which each model was of greatest value. In the validation cohort, the overall postoperative UTI prevalence was 11% (95% CI 8, 15). The original model had low discriminatory ability in the validation cohort (C-index = 0.53) and was miscalibrated. Recalibration alone did not improve model performance. The model coefficients and intercepts were updated to the validation cohort after adding the new predictors of sling procedure, HT, age, recurrent UTI history, postoperative bladder drainage, ASA score, and hysterectomy. Model discrimination improved (C-index = 0.61, 95% CI 0.52,0.68) with Brier score of 0.1 and moderate calibration (Figure 1). Decision curve analysis demonstrated benefit with treatment thresholds of 5 to 45% (Figure 2). The original postoperative UTI model required updating to achieve acceptable discrimination and calibration in a cohort of women undergoing urogynecologic surgery. This updated model can be used to identify individuals at high-risk for postoperative UTI and may facilitate targeted prevention strategies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

"Omics" Technologies - What Have They Told Us About Uropathogenic Escherichia coli Fitness and Virulence During Urinary Tract Infection?

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infection (UTI), a widespread infectious disease of great impact on human health. This is further emphasized by the rapidly increase in antimicrobial resistance in UPEC, which compromises UTI treatment. UPEC biology is highly complex since uropathogens must adopt extracellular and intracellular lifestyles and adapt to different niches in the host. In this context, the implementation of forefront ‘omics’ technologies has provided substantial insight into the understanding of UPEC pathogenesis, which has opened the doors for new therapeutics and prophylactics discovery programs. Thus, ‘omics’ technologies applied to studies of UPEC during UTI, or in models of UTI, have revealed extensive lists of factors that are important for the ability of UPEC to cause disease. The multitude of large ‘omics’ datasets that have been generated calls for scrutinized analysis of specific factors that may be of interest for further development of novel treatment strategies. In this review, we describe main UPEC determinants involved in UTI as estimated by ‘omics’ studies, and we compare prediction of factors across the different ‘omics’ technologies, with a focus on those that have been confirmed to be relevant under UTI-related conditions. We also discuss current challenges and future perspectives regarding analysis of data to provide an overview and better understanding of UPEC mechanisms involved in pathogenesis which should assist in the selection of target sites for future prophylaxis and treatment.

Risk-stratification in febrile infants 29 to 60\u2009days old: a cost-effectiveness analysis

BackgroundMultiple clinical prediction rules have been published to risk-stratify febrile infants ≤60 days of age for serious bacterial infections (SBI), which is present in 8-13% of infants. We evaluate the cost-effectiveness of strategies to identify infants with SBI in the emergency department.MethodsWe developed a Markov decision model to estimate outcomes in well-appearing, febrile term infants, using the following strategies: Boston, Rochester, Philadelphia, Modified Philadelphia, Pediatric Emergency Care Applied Research Network (PECARN), Step-by-Step, Aronson, and clinical suspicion. Infants were categorized as low risk or not low risk using each strategy. Simulated cohorts were followed for 1 year from a healthcare perspective. Our primary model focused on bacteremia, with secondary models for urinary tract infection and bacterial meningitis. One-way, structural, and probabilistic sensitivity analyses were performed. The main outcomes were SBI correctly diagnosed and incremental cost per quality-adjusted life-year (QALY) gained.ResultsIn the bacteremia model, the PECARN strategy was the least expensive strategy ($3671, 0.779 QALYs). The Boston strategy was the most cost-effective strategy and cost $9799/QALY gained. All other strategies were less effective and more costly. Despite low initial costs, clinical suspicion was among the most expensive and least effective strategies. Results were sensitive to the specificity of selected strategies. In probabilistic sensitivity analyses, the Boston strategy was most likely to be favored at a willingness-to-pay threshold of $100,000/QALY. In the urinary tract infection model, PECARN was preferred compared to other strategies and the Boston strategy was preferred in the bacterial meningitis model.ConclusionsThe Boston clinical prediction rule offers an economically reasonable strategy compared to alternatives for identification of SBI.

A Biomimetic Nonantibiotic Nanoplatform for Low-Temperature Photothermal Treatment of Urinary Tract Infections Caused by Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) remain a matter of concern, as the clinical use of multiple antibiotics induces antibiotic resistance in bacteria, resulting in the failure of treatments. Despite the emergence of anti-adhesion strategies that can prevent the development of bacterial drug resistance, these strategies are mainly used for disease prevention rather than effective treatment. Photothermal therapy (PTT) has emerged as an efficient alternative for the elimination of bacteria. Nevertheless, high local temperatures related to PTT probably cause damage to surrounding healthy tissue. Herein, a biomimetic nonantibiotic nanoplatform for low-temperature photothermal treatment of UTIs is developed. The nanoplatform comprises polydopamine (PDA) photothermal core and biphenyl mannoside (Man) shell with multivalent high-affinity to UPEC. Scanning electron microscope (SEM) shows PDA-Man possessed ultra-strong targeting binding ability toward UPEC. It is the fact that this impulse UPEC to form a large bacterial cluster. Consequently, the high photothermal energy of the PDA-Man appears predominantly in the affected bacterial area, while the overall environment remains at a low temperature. The fabricated nanoplatform shows excellent photothermal bactericidal effects, approximately 100% in a UTI model. Overall, this low-temperature photothermal nanoplatform provides an appropriate strategy for the elimination of bacteria in clinical applications.

Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria.

The prevalence of multidrug-resistant bacteria has been increasing rapidly worldwide, a trend that poses great risk to human and animal health and creates urgent need for pharmaceutical and nonpharmaceutical approaches to stop the spread of disease due to antimicrobial resistance. Here, we found that alanine, aspartate, and glutamate metabolism was inactivated, and glutamine was repressed in multidrug-resistant uropathogenic Escherichia coli using a comparative metabolomics approach. Exogenous glutamine promoted β-lactam–, aminoglycoside-, quinolone-, and tetracycline-induced killing of uropathogenic E. coli and potentiated ampicillin to eliminate multidrug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella peneumoniae, Edwardsiella tarda, Vibrio alginolyticus, and Vibrio parahaemolyticus. Glutamine-potentiated ampicillin-mediated killing was effective against biofilms of these bacteria in a mouse urinary tract infection model and against systemic infection caused by E. coli, P. aeruginosa, A. baumannii, or K. peneumoniae in a mouse model. Exogenous glutamine stimulated influx of ampicillin, leading to the accumulation of intracellular antibiotic concentrations that exceeded the amount tolerated by the multidrug-resistant bacteria. Furthermore, we demonstrated that exogenous glutamine promoted the biosynthesis of nucleosides including inosine, which in turn interacted with CpxA/CpxR and up-regulated OmpF. We validated the physiological relevance of the mechanism by showing that loss of purF, purH, cpxA, or ompF elevated antibiotic resistance in antibiotic-sensitive strains. In addition, glutamine retarded the development of ampicillin resistance. These results may facilitate future development of effective approaches for preventing or managing chronic, multidrug-resistant bacterial infections, bacterial persistence, and difficult-to-treat bacterial biofilms.

TP0586532, a non-hydroxamate LpxC inhibitor, has in vitro and in vivo antibacterial activities against Enterobacteriaceae.

The emergence of multi-drug resistant pathogenic bacteria, especially Gram-negative bacteria, is a worldwide health problem. New antibiotics directed at previously unexplored targets are urgently needed to overcome resistance to existing antibiotic classes. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an attractive target for a new antibacterial agent. Although a number of LpxC inhibitors have been identified, none have been approved as antibacterial agents. These LpxC inhibitors contain a hydroxamate moiety, which is a robust zinc ion chelator. The nonspecific inhibition of metalloenzymes through zinc ion chelation is one of possibilities leading to unwanted side effects. Herein, we report that TP0586532, a non-hydroxamate LpxC inhibitor, has a broad spectrum of antibacterial activity against carbapenem-resistant Enterobacteriaceae. The MIC90 of TP0586532 against clinical isolates of carbapenem-resistant Klebsiella pneumoniae was 4 μg ml-1. TP0586532 also showed an in vivo efficacy against murine systemic, urinary tract and lung infection models caused by meropenem- or ciprofloxacin-resistant strains. The estimated maximum unbound plasma concentration value at the effective dose of TP0586532 in murine infection models was around 13 μg ml-1. TP0586532 is predicted to exhibit a in vivo efficacy without cardiovascular toxicity and showed the potential of non-hydroxamate LpxC inhibitors as antibacterial agents against carbapenem-resistant Enterobacteriaceae.

Mechanistic Studies of Antibiotic Adjuvants Reducing Kidney's Bacterial Loads upon Systemic Monotherapy.

We describe the design and attributes of a linear pentapeptide-like derivative (C14(ω5)OOc10O) screened for its ability to elicit bactericidal competences of plasma constituents against Gram-negative bacteria (GNB). In simpler culture media, the lipopeptide revealed high aptitudes to sensitize resilient GNB to hydrophobic and/or efflux-substrate antibiotics, whereas in their absence, C14(ω5)OOc10O only briefly delayed bacterial proliferation. Instead, at low micromolar concentrations, the lipopeptide has rapidly lowered bacterial proton and ATP levels, although significantly less than upon treatment with its bactericidal analog. Mechanistic studies support a two-step scenario providing a plausible explanation for the lipopeptide’s biological outcomes against GNB: initially, C14(ω5)OOc10O permeabilizes the outer membrane similarly to polymyxin B, albeit in a manner not necessitating as much LPS-binding affinity. Subsequently, C14(ω5)OOc10O would interact with the inner membrane gently yet intensively enough to restrain membrane-protein functions such as drug efflux and/or ATP generation, while averting the harsher inner membrane perturbations that mediate the fatal outcome associated with bactericidal peers. Preliminary in vivo studies where skin wound infections were introduced in mice, revealed a significant efficacy in affecting bacterial viability upon topical treatment with creams containing C14(ω5)OOc10O, whereas synergistic combination therapies were able to secure the pathogen’s eradication. Further, capitalizing on the finding that C14(ω5)OOc10O plasma-potentiating concentrations were attainable in mice blood at sub-maximal tolerated doses, we used a urinary tract infection model to acquire evidence for the lipopeptide’s systemic capacity to reduce the kidney’s bacterial loads. Collectively, the data establish the role of C14(ω5)OOc10O as a compelling antibacterial potentiator and suggest its drug-like potential.

Clinical exposure-response relationship of cefepime/taniborbactam against Gram-negative organisms in the murine complicated urinary tract infection model.

Complicated urinary tract infections (cUTIs) are frequently encountered in hospitals and ICUs. Increasingly, the causative pathogens harbour enzymatic resistance mechanisms. Taniborbactam is a novel β-lactamase inhibitor with activity against Ambler class A, B, C and D β-lactamases. Herein, we assessed the efficacy of cefepime alone and the combination cefepime/taniborbactam in a neutropenic murine cUTI model. Eighteen cefepime-resistant clinical isolates (9 Enterobacterales, 3 Pseudomonas aeruginosa and 6 Stenotrophomonas maltophilia; cefepime MIC = 32 to >512 mg/L) were assessed. Cefepime/taniborbactam MICs ranged from 0.06 to 128 mg/L. Human-simulated plasma regimens (HSRs) of cefepime alone and in combination with taniborbactam were developed in the murine cUTI model. The efficacy of cefepime HSR and cefepime/taniborbactam HSR was determined as the change in log10 cfu/kidney at 48 h compared with 48 h controls. Mean ± SD initial bacterial burden was 5.66 ± 0.56 log10 cfu/kidney, which increased to 9.05 ± 0.39 log10 cfu/kidney at 48 h. The cefepime HSR was ineffective, as bacterial burden was similar to untreated controls (-0.14 ± 0.40 change in log10 cfu/kidney). In contrast, cefepime/taniborbactam exhibited substantial killing, with log10 cfu/kidney changes of -5.48 ± 1.3, -4.79 ± 0.3 and -5.04 ± 0.7 for ESBL/AmpC-, KPC- and OXA-48-harbouring Enterobacterales, respectively. Cefepime/taniborbactam also exhibited robust killing of P. aeruginosa (-6.5 ± 0.26) and S. maltophilia (-5.66 ± 0.71). Humanized exposures of cefepime/taniborbactam achieved robust killing of Enterobacterales, P. aeruginosa and S. maltophilia harbouring ESBL, AmpC, KPC and/or OXA-48. These data support the role of cefepime/taniborbactam for cUTI treatment for cefepime/taniborbactam MICs up to 32 mg/L.

A Novel Phage Cocktail Therapy of the Urinary Tract Infection in a Mouse Model.

Escherichia coli (E. coli) is a major bacterial pathogen associated with many cases of serious infections, such as urinary tract infections (UTI) and meningitis intestinal. The rapid emergence of antimicrobial multidrug-resistant bacteria occurring worldwide has been attributed to the overuse of antibiotics. Alternative strategies must be developed to overcome antibiotic resistance. A promising alternative for the treatment of infections is the use of phages as antibacterial agents. A total of 90 female albino mice were randomly divided into three groups (n=30) and used for the induction of UTI. The animals were acclimatized in their cages for 24 h before inoculation and allowed to access chow and water freely. For UTI induction, the peri-urethral area was sterilized with 70% ethanol, and bacterial inoculation was then injected into the bladder through the urethra using a 24-gauge sterile Teflon catheter with an outer diameter of 0.7 mm and length of 19 mm. A single phage and a phage cocktail preparation have been evaluated for their therapeutic activity in the mouse model of chronic UTI induced by transurethral injection of two isolates of the uropathogenic E. coli 8 and E. coli 302. The results of the transurethral and intra-peritoneal injection of phage(s) that prepared on day 10 after the establishment of the mouse chronic model showed no effect of a single phage PEC80 in the treatment of UTI, whereas both administration routes of the phage cocktail preparation resulted in the clearance of bacteria from mice urine and homogenates of the urinary bladders and kidneys of the sacrificed mice after 24 h following the administration of phage cocktail dose. The high activity of the phage cocktail in the treatment of mouse chronic model of UTI is attributed to the broader host range of the phage cocktail, compared to the very narrow host range of the phage PEC80. It is concluded that the phage therapy by using phage preparations as the 25 phages cocktail evaluated in this study is a highly promising and potential alternative therapy for human UTIs.

Interleukin-6 mediates delirium-like phenotypes in a murine model of urinary tract infection

BackgroundUrinary tract infection (UTI) is frequently implicated as a precipitant of delirium, which refers to an acute confusional state that is associated with high mortality, increased length of stay, and long-term cognitive decline. The pathogenesis of delirium is thought to involve cytokine-mediated neuronal dysfunction of the frontal cortex and hippocampus. We hypothesized that systemic IL-6 inhibition would mitigate delirium-like phenotypes in a mouse model of UTI.MethodsC57/BL6 mice were randomized to either: (1) non-UTI control, (2) UTI, and (3) UTI + anti-IL-6 antibody. UTI was induced by transurethral inoculation of 1 × 108Escherichia coli. Frontal cortex and hippocampus-mediated behaviors were evaluated using functional testing and corresponding structural changes were evaluated via quantification of neuronal cleaved caspase-3 (CC3) by immunohistochemistry and western blot. IL-6 in the brain and plasma were evaluated using immunohistochemistry, ELISA, and RT-PCR.ResultsCompared to non-UTI control mice, mice with UTI demonstrated significantly greater impairments in frontal and hippocampus-mediated behaviors, specifically increased thigmotaxis in Open Field (p < 0.05) and reduced spontaneous alternations in Y-maze (p < 0.01), while treatment of UTI mice with systemic anti-IL-6 fully reversed these functional impairments. These behavioral impairments correlated with frontal and hippocampal neuronal CC3 changes, with significantly increased frontal and hippocampal CC3 in UTI mice compared to non-UTI controls (p < 0.0001), and full reversal of UTI-induced CC3 neuronal changes following treatment with systemic anti-IL-6 antibody (p < 0.0001). Plasma IL-6 was significantly elevated in UTI mice compared to non-UTI controls (p < 0.01) and there were positive and significant correlations between plasma IL-6 and frontal CC3 (r2 = 0.5087/p = 0.0028) and frontal IL-6 and CC3 (r2 = 0.2653, p < 0.0001).ConclusionsThese data provide evidence for a role for IL-6 in mediating delirium-like phenotypes in a mouse model of UTI. These findings provide pre-clinical justification for clinical investigations of IL-6 inhibitors to treat UTI-induced delirium.

Investigating Candida glabrata Urinary Tract Infections (UTIs) in Mice Using Bioluminescence Imaging.

Urinary tract infections (UTIs) are quite common and mainly caused by bacteria such as Escherichia coli. However, when patients have urinary catheters, fungal infections comprise up to 15% of these types of infections. Moreover, fungal UTIs have a high mortality, due to rapid spreading of the fungi to the kidneys. Most fungal UTIs are caused by Candida species, among which Candida albicans and Candida glabrata are the most common. C. glabrata is an opportunistic pathogenic yeast, phylogenetically quite close to Saccharomyces cerevisiae. Even though it is commonly isolated from the urinary tract and rapidly acquires resistance to antifungals, its pathogenesis has not been studied extensively in vivo. In vivo studies require high numbers of animals, which can be overcome by the use of non-invasive imaging tools. One such tool, bioluminescence imaging, has been used successfully to study different types of C. albicans infections. For C. glabrata, only biofilms on subcutaneously implanted catheters have been imaged using this tool. In this work, we investigated the progression of C. glabrata UTIs from the bladder to the kidneys and the spleen. Furthermore, we optimized expression of a red-shifted firefly luciferase in C. glabrata for in vivo use. We propose the first animal model using bioluminescence imaging to visualize C. glabrata in mouse tissues. Additionally, this UTI model can be used to monitor antifungal activity in vivo over time.

Escherichia coli type-1 fimbriae are critical to overcome initial bottlenecks of infection upon low-dose inoculation in a porcine model of cystitis.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models’ natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

NRF2 promotes urothelial cell response to bacterial infection by regulating reactive oxygen species and RAB27B expression.

Uropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) by invading urothelial cells. In response, the host mounts an inflammatory response to expel bacteria. Here, we show that the NF-E2-related factor 2 (NRF2) pathway is activated in response to UPEC-triggered reactive oxygen species (ROS) production. We demonstrate the molecular sequence of events wherein NRF2 activation in urothelial cells reduces ROS production, inflammation, and cell death, promotes UPEC expulsion, and reduces the bacterial load. In contrast, loss of NRF2 leads to increased ROS production, bacterial burden, and inflammation, both invitro and invivo. NRF2 promotes UPEC expulsion by regulating transcription of the RAB-GTPase RAB27B. Finally, dimethyl fumarate, a US Food and Administration-approved NRF2 inducer, reduces the inflammatory response, increases RAB27B expression, and lowers bacterial burden in urothelial cells and in a mouse UTI model. Our findings elucidate mechanisms underlying the host response to UPEC and provide a potential strategy to combat UTIs.

Bis-molybdopterin guanine dinucleotide modulates hemolysin expression under anaerobiosis and contributes to fitness in vivo in uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). Successful urinary tract colonization requires appropriate expression of virulence factors in response to host environmental cues, such as limited oxygen and iron availability. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Previously, we showed that hemolysin expression is enhanced under anaerobic conditions; however, the genetic basis and regulatory mechanisms involved remain undefined. Here, a transposon-based forward screen identified bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) biosynthesis as an important factor for a full transcription of hemolysin under anaerobiosis but not under aerobiosis. bis-MGD positively influences hemolysin transcription via c3566-c3568, an operon immediately upstream of and cotranscribed with hlyCABD. Furthermore, suppressor mutation analysis identified the nitrogen regulator NtrC as a direct repressor of c3566-c3568-hlyCABD expression, and intact bis-MGD biosynthesis downregulated ntrC expression, thus at least partially explaining the positive role of bis-MGD in modulating hemolysin expression. Finally, bis-MGD is involved in hemolysin-mediated uroepithelial cell death and contributes to the competitive fitness of UPEC in a murine model of UTI. Collectively, our data establish that bis-MGD biosynthesis plays a crucial role in UPEC fitness in vivo, thus providing a potential target for combatting UTIs.