A human urothelial microtissue model reveals shared colonization and survival strategies between uropathogens and commensals.

Urinary tract infection is among the most common infections worldwide, typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial species in a human urothelial microtissue model exhibiting full stratification, differentiation, innate epithelial responses, and urine tolerance. Several uropathogens invaded intracellularly, but also commensal Escherichia coli, suggesting that invasion is a shared survival strategy, not solely a virulence hallmark. The E. coli adhesin FimH was required for intracellular bacterial community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives), and hijacking of exfoliating cells, while biofilm-like aggregates were formed mainly with Pseudomonas and Proteus. Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not commensals, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the microtissue, cell, and molecular level.

Drug delivery strategies for antibiofilm therapy.

Although new antibiofilm agents have been developed to prevent and eliminate pathogenic biofilms, their widespread clinical use is hindered by poor biocompatibility and bioavailability, unspecific interactions and insufficient local concentrations. The development of innovative drug delivery strategies can facilitate penetration of antimicrobials through biofilms, promote drug dispersal and synergistic bactericidal effects, and provide novel paradigms for clinical application. In this Review, we discuss the potential benefits of such emerging techniques for improving the clinical efficacy of antibiofilm agents, as well as highlighting the existing limitations and future prospects for these therapies in the clinic.

An immunoresponsive three-dimensional urine-tolerant human urothelial model to study urinary tract infection.

Introduction: Murine models of urinary tract infection (UTI) have improved our understanding of host-pathogen interactions. However, given differences between rodent and human bladders which may modulate host and bacterial response, including certain biomarkers, urothelial thickness and the concentration of urine, the development of new human-based models is important to complement mouse studies and to provide a more complete picture of UTI in patients. Methods: We originally developed a human urothelial three-dimensional (3D) model which was urine tolerant and demonstrated several urothelial biomarkers, but it only achieved human thickness in heterogenous, multi-layered zones and did not demonstrate the comprehensive differentiation status needed to achieve barrier function. We optimised this model by altering a variety of conditions and validated it with microscopy, flow cytometry, transepithelial electrical resistance and FITC-dextran permeability assays to confirm tissue architecture, barrier integrity and response to bacterial infection. Results: We achieved an improved 3D urine-tolerant human urothelial model (3D-UHU), which after 18-20 days of growth, stratified uniformly to 7-8 layers comprised of the three expected, distinct human cell types. The apical surface differentiated into large, CD227+ umbrella-like cells expressing uroplakin-1A, II, III, and cytokeratin 20, all of which are important terminal differentiation markers, and a glycosaminoglycan layer. Below this layer, several layers of intermediate cells were present, with a single underlying layer of CD271+ basal cells. The apical surface also expressed E-cadherin, ZO-1, claudin-1 and -3, and the model possessed good barrier function. Infection with both Gram-negative and Gram-positive bacterial classes elicited elevated levels of pro-inflammatory cytokines and chemokines characteristic of urinary tract infection in humans and caused a decrease in barrier function. Discussion: Taken together, 3D-UHU holds promise for studying host-pathogen interactions and host urothelial immune response.

Effective Treatments of UTI-Is Intravesical Therapy the Future?

Urinary tract infection (UTI) afflicts millions of patients globally each year. While the majority of UTIs are successfully treated with orally administered antibiotics, the impact of oral antibiotics on the host microbiota is under close research scrutiny and the potential for dysbiosis is a cause for concern. Optimal treatment of UTI relies upon the selection of an agent which displays appropriate pharmacokinetic-pharmacodynamic (PK-PD) properties that will deliver appropriately high concentrations in the urinary tract after oral administration. Alternatively, high local concentrations of antibiotic at the urothelial surface can be achieved by direct instillation into the urinary tract. For antibiotics with the appropriate physicochemical properties, this can be of critical importance in cases for which an intracellular urothelial bacterial reservoir is suspected. In this review, we summarise the underpinning biopharmaceutical barriers to effective treatment of UTI and provide an overview of the evidence for the deployment of the intravesical administration route for antibiotics.

Application of Various Techniques to Gain Insights Into the Complex Urinary Tract Microbial Communities of Renal Transplant Recipients.

Urinary tract infections (UTIs) are prevalent in renal transplant (RTX) recipients and associated with worse outcomes. Early detection by sensitive diagnostic tests and appropriate treatment strategies in this cohort is therefore crucial, but evidence has shown that current methods may miss genuine infections. Research has shed light on the urinary tract microbial ecology of healthy individuals and nontransplant patients with UTI, but information on the RTx cohort is scant. We conducted a cross-sectional study to (i) compare the gold standard diagnostic culture with alternative techniques and (ii) characterize RTx patient urinary microbial communities. Methods: Midstream urine specimens were collected from 51 RTx patients attending a renal transplant clinic and 27 asymptomatic controls. Urinary microscopy, dipstick, and routine culture were performed. To improve sensitivity of microbial detection, we cultured the urinary cell sediment and performed 16S rRNA gene sequencing on urine. Uroplakin-positive urothelial cells shed in urine were analyzed by immunofluorescence staining for any bacterial association. Results: Sediment culture and 16S rRNA sequencing confirmed detection deficiencies of diagnostic culture and revealed differences in the urobiomes of RTx patients and controls. Specifically, Gardnerella, Escherichia, and Lactobacillus were most abundant in patients, whereas Lactobacillus, Streptococcus, and Gardnerella were most abundant in controls. The application of both culture and sequencing provided a more nuanced view of the urinary microbial communities. Conclusions: This study provides insight into the potential problems of diagnostic culture within RTx patients and sheds light on their urinary microbial inhabitants. Further work may identify key microbial signatures and facilitate the development of better tools for UTI detection within this cohort, which could allow targeted intervention before an infection leads to serious consequences. http://links.lww.com/TXD/A479.

The urothelium: a multi-faceted barrier against a harsh environment.

All mucosal surfaces must deal with the challenge of exposure to the outside world. The urothelium is a highly specialized layer of stratified epithelial cells lining the inner surface of the urinary bladder, a gruelling environment involving significant stretch forces, osmotic and hydrostatic pressures, toxic substances, and microbial invasion. The urinary bladder plays an important barrier role and allows the accommodation and expulsion of large volumes of urine without permitting urine components to diffuse across. The urothelium is made up of three cell types, basal, intermediate, and umbrella cells, whose specialized functions aid in the bladder's mission. In this review, we summarize the recent insights into urothelial structure, function, development, regeneration, and in particular the role of umbrella cells in barrier formation and maintenance. We briefly review diseases which involve the bladder and discuss current human urothelial in vitro models as a complement to traditional animal studies.

Severe Acute Respiratory Syndrome Type 2-Causing Coronavirus: Variants and Preventive Strategies.

COVID-19 vaccines have constituted a substantial scientific leap in countering severe acute respiratory syndrome type 2-causing coronavirus (SARS-CoV-2), and worldwide implementation of vaccination programs has significantly contributed to the global pandemic effort by saving many lives. However, the continuous evolution of the SARS-CoV-2 viral genome has resulted in different variants with a diverse range of mutations, some with enhanced virulence compared with previous lineages. Such variants are still a great concern as they have the potential to reduce vaccine efficacy and increase the viral transmission rate. This review summarizes the significant variants of SARS-CoV-2 encountered to date (December 2021) and discusses a spectrum of possible preventive strategies, with an emphasis on physical and materials science.

Recurrent Urinary Tract Infection: A Mystery in Search of Better Model Systems.

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide but are significantly understudied. Uropathogenic E. coli (UPEC) accounts for a significant proportion of UTI, but a large number of other species can infect the urinary tract, each of which will have unique host-pathogen interactions with the bladder environment. Given the substantial economic burden of UTI and its increasing antibiotic resistance, there is an urgent need to better understand UTI pathophysiology - especially its tendency to relapse and recur. Most models developed to date use murine infection; few human-relevant models exist. Of these, the majority of in vitro UTI models have utilized cells in static culture, but UTI needs to be studied in the context of the unique aspects of the bladder's biophysical environment (e.g., tissue architecture, urine, fluid flow, and stretch). In this review, we summarize the complexities of recurrent UTI, critically assess current infection models and discuss potential improvements. More advanced human cell-based in vitro models have the potential to enable a better understanding of the etiology of UTI disease and to provide a complementary platform alongside animals for drug screening and the search for better treatments.

Novel antibiotic-loaded particles conferring eradication of deep tissue bacterial reservoirs for the treatment of chronic urinary tract infection.

A significant proportion of urinary tract infection (UTI) patients experience recurrent episodes, due to deep tissue infection and treatment-resistant bacterial reservoirs. Direct bladder instillation of antibiotics has proved disappointing in treating UTI, likely due to the failure of infused antibiotics to penetrate the bladder epithelium and accumulate to high enough levels to kill intracellular bacteria. This work investigates the use of nitrofurantoin loaded poly(lactic-co-glycolic acid) (PLGA) particles to improve delivery to intracellular targets for the treatment of chronic UTI. Using electrohydrodynamic atomisation, we produced particles with an average diameter of 2.8 µm. In broth culture experiments, the biodegradable particles were effective against a number of UTI-relevant bacterial strains. Dye-loaded particles demonstrated that intracellular delivery was achieved in all cells in 2D cultures of a human bladder epithelial progenitor cell line in a dose-dependent manner, achieving far higher efficiency and concentration than equivalent quantities of free drug. Time-lapse video microscopy confirmed that delivery occurred within 30 min of administration, to 100% of cells. Moreover, the particles were able to deliver the drug to cells through multiple layers of a 3D human bladder organoid model causing minimal cell toxicity, displaying superior killing of bacterial reservoirs harboured within bladder cells compared with unencapsulated drug. The particles were also able to kill bacterial biofilms more effectively than the free drug. These results illustrate the potential for using antibiotic-loaded microparticles to effectively treat chronic UTIs. Such a delivery method could be extrapolated to other clinical indications where robust intracellular delivery is required, such as oncology and gene therapy.

The great host-pathogen war: U.K. Cellular microbiology meeting 2020.

In 2019 we started a new annual meeting, aimed at bringing together researchers from across the United Kingdom studying cellular microbiology and the cell biology of host-pathogen interactions. In contrast to large glamourous meetings, featuring the great and the good from across the world, we wanted to create a forum for early career researchers to present their work and enjoy lively discussion. In particular, we hope that focussing on making the meeting accessible, affordable, and informal would help integrate and build the U.K. community working on this exciting topic.

Effect of Environment on the Evolutionary Trajectories and Growth Characteristics of Antibiotic-Resistant Escherichia coli Mutants.

The fitness cost to bacteria of acquisition of resistance determinants is critically under-investigated, and the identification and exploitation of these fitness costs may lead to novel therapeutic strategies that prevent the emergence of antimicrobial resistance. Here we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how the artificial environments utilized in studies of bacterial fitness could affect the emergence of resistance and associated fitness costs. Further, we explored the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-h exposure to sub-inhibitory concentrations of AMC in either M9, ISO, or LB, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine, and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This preliminary study highlights the risks in relying on a single culture protocol as a model system to predict the behavior and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes.

The UK cellular microbiology network: Exploring the host-bacterial interface.

The UK Cellular Microbiology Network held its inaugural conference in February 2019. This stimulating day of scientific exchange will be the first of many, its organisers hope.

Reassessment of Routine Midstream Culture in Diagnosis of Urinary Tract Infection.

Midstream urine (MSU) culture remains the gold standard diagnostic test for confirming urinary tract infection (UTI). We previously showed that patients with chronic lower urinary tract symptoms (LUTS) below the diagnostic cutoff on MSU culture may still harbor bacterial infection and that their antibiotic treatment was associated with symptom resolution. Here, we evaluated the results of the United Kingdom's MSU culture in symptomatic patients and controls. Next, we compared the bacterial enrichment capabilities of the MSU culture with those of a 50-µl uncentrifuged culture, a 30-ml centrifuged sediment culture, and 16S rRNA gene sequencing. This study was conducted on urine specimens from 33 LUTS patients attending their first clinical appointment (mean age, 48.7 years; standard deviation [SD], 16.5 years), 30 LUTS patients on treatment (mean age, 47.8 years; SD, 16.5 years) whose symptoms had relapsed, and 29 asymptomatic controls (mean age, 40.7 years, SD, 15.7 years). We showed that the routine MSU culture, adopting the UK interpretation criteria tailored to acute UTI, failed to detect a variety of bacterial species, including recognized uropathogens. Moreover, the diagnostic MSU culture was unable to discriminate between patients and controls. In contrast, genomic analysis of urine enriched by centrifugation discriminated between the groups, generating a more accurate understanding of species richness. In conclusion, the United Kingdom's MSU protocol misses a significant proportion of bacteria, which include recognized uropathogens, and may be unsuitable for excluding UTI in patients with LUTS.

Cross-over data supporting long-term antibiotic treatment in patients with painful lower urinary tract symptoms, pyuria and negative urinalysis.

PURPOSE: To measure the effects of an unplanned, sudden cessation of treatment in an unselected group of patients with chronic painful LUTS managed with protracted antimicrobial treatment and to report these observational data collected from a cross-over process. MATERIALS AND METHODS: The imposition of a guideline resulted in the immediate cessation of antibiotic treatment in a cohort of patients with chronic painful LUTS and microscopic pyuria. Patients were assessed before treatment withdrawal, whilst off treatment, and following reinstatement. Outcome measures included a validated symptom score, microscopic enumeration of urinary white cells and uroepithelial cells, and routine urine culture. RESULTS: These patients had reported treatment-resistant, painful LUTS for a mean of 6.5 years before treatment at this centre. Treatment was stopped in 221 patients (female = 210; male = 11; mean age = 56 years; SD = 17.81). Sixty-six per cent of women were post-menopausal. After unplanned treatment cessation, 199 patients (90%; female = 188; male = 9) reported deterioration. Eleven patients required hospital care in association with disease recurrence, including acute urinary tract infection (UTI) and urosepsis. Symptom scores increased after cessation and recovered on reinitiating treatment (F = 33; df = 2; p < 0.001). Urinary leucocyte (F = 3.7; df = 2; p = 0.026) and urothelial cells counts mirrored symptomatic changes (F = 6.0; df = 2; p = 0.003). Routine urine culture results did not reflect changes in disease status. CONCLUSION: These data support the hypothesis that treating painful LUTS associated with pyuria with long-term antimicrobial courses, despite negative urine culture, is effective. The microscopy of fresh unspun, unstained urine to count white cells and epithelial cells offers a valid method of monitoring disease. An unplanned cessation of antibiotic therapy produced a resurgence of symptoms and lower urinary tract inflammation in patients with chronic LUTS, supporting an infective aetiology below the level of routine detection.

Recalcitrant chronic bladder pain and recurrent cystitis but negative urinalysis: What should we do?

PURPOSE: Lower urinary tract symptoms (LUTS) may be associated with chronic urinary tract infection (UTI) undetected by routine diagnostic tests. Antimicrobial therapy might confer benefit for these patients. MATERIALS AND METHODS: Over 10 years, we treated patients with chronic LUTS. Pyuria was adopted as the principal biomarker of infection. Urinary leucocyte counts were recorded from microscopy of fresh midstream urine (MSU) samples. Antibiotics were prescribed and the prescription adjusted to achieve a measurable clinical response and a reduction in pyuria. RESULTS: We treated 624 women [mean age = 53.4 years; standard deviation (SD) = 18] with chronic LUTS and pyuria. Mean duration of symptoms prior to presentation was 6.5 years. Only 16% of MSU cultures submitted were positive (≥105 cfu ml-1). Mean treatment length was 383 days [SD = 347; 95% confidence interval (CI) = 337-428]. Treatment was associated with a reduction in total LUTS (F = 98; p = 0.0001), 24-h frequency (F = 75; p = 0.0001), urinary urgency (F = 90; p = 0.0001), lower urinary tract pain (F = 108; p = 0.0001), voiding symptoms (F = 10; p = 0.002), and pyuria (F = 15.4; p = 0.0001). Full-dose first-generation antibiotics for UTI, such as cefalexin, nitrofurantoin, or trimethoprim, were combined with methenamine hippurate. We recorded 475 adverse events (AEs) during 273,762 treatment days. There was only one serious adverse event (SAE). We observed no increase in the proportion of resistant bacterial isolates. CONCLUSION: This large case series demonstrates that patients with chronic LUTS and pyuria experience symptom regression and a reduction in urinary tract inflammation associated with antimicrobial therapy. Disease regression was achieved with a low frequency of AEs. These results provide preliminary data to inform a future randomized controlled trial (RCT).

A urine-dependent human urothelial organoid offers a potential alternative to rodent models of infection.

Murine models describe a defined host/pathogen interaction for urinary tract infection, but human cell studies are scant. Although recent human urothelial organoid models are promising, none demonstrate long-term tolerance to urine, the natural substrate of the tissue and of the uropathogens that live there. We developed a novel human organoid from progenitor cells which demonstrates key structural hallmarks and biomarkers of the urothelium. After three weeks of transwell culture with 100% urine at the apical interface, the organoid stratified into multiple layers. The apical surface differentiated into enlarged and flattened umbrella-like cells bearing characteristic tight junctions, structures resembling asymmetric unit membrane plaques, and a glycosaminoglycan layer. The apical cells also expressed cytokeratin-20, a spatial feature of the mammalian urothelium. Urine itself was necessary for full development, and undifferentiated cells were urine-tolerant despite the lack of membrane plaques and a glycosaminoglycan layer. Infection with Enterococcus faecalis revealed the expected invasive outcome, including urothelial sloughing and the formation of intracellular colonies similar to those previously observed in patient cells. This new biomimetic model could help illuminate invasive behaviours of uropathogens, and serve as a reproducible test bed for disease formation, treatment and resolution in patients.

Evolution of Communities in the Medical Sciences: Evidence from the Medical Words Network.

BACKGROUND: Classification of medical sciences into its sub-branches is crucial for optimum administration of healthcare and specialty training. Due to the rapid and continuous evolution of medical sciences, development of unbiased tools for monitoring the evolution of medical disciplines is required. METHODOLOGY/PRINCIPAL FINDINGS: Network analysis was used to explore how the medical sciences have evolved between 1980 and 2015 based on the shared words contained in more than 9 million PubMed abstracts. The k-clique percolation method was used to extract local research communities within the network. Analysis of the shared vocabulary in research papers reflects the trends of collaboration and splintering among different disciplines in medicine. Our model identifies distinct communities within each discipline that preferentially collaborate with other communities within other domains of specialty, and overturns some common perceptions. CONCLUSIONS/SIGNIFICANCE: Our analysis provides a tool to assess growth, merging, splitting and contraction of research communities and can thereby serve as a guide to inform policymakers about funding and training in healthcare.

Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms.

BACKGROUND: Adenosine-5'-triphosphate (ATP) is a neurotransmitter and inflammatory cytokine implicated in the pathophysiology of lower urinary tract disease. ATP additionally reflects microbial biomass thus has potential as a surrogate marker of urinary tract infection (UTI). The optimum clinical sampling method for ATP urinalysis has not been established. We tested the potential of urinary ATP in the assessment of lower urinary tract symptoms, infection and inflammation, and validated sampling methods for clinical practice. METHODS: A prospective, blinded, cross-sectional observational study of adult patients presenting with lower urinary tract symptoms (LUTS) and asymptomatic controls, was conducted between October 2009 and October 2012. Urinary ATP was assayed by a luciferin-luciferase method, pyuria counted by microscopy of fresh unspun urine and symptoms assessed using validated questionnaires. The sample collection, storage and processing methods were also validated. RESULTS: 75 controls and 340 patients with LUTS were grouped as without pyuria (n = 100), pyuria 1-9 wbc µl(-1) (n = 120) and pyuria ≥10 wbc µl(-1) (n = 120). Urinary ATP was higher in association with female gender, voiding symptoms, pyuria greater than 10 wbc µl(-1) and negative MSU culture. ROC curve analysis showed no evidence of diagnostic test potential. The urinary ATP signal decayed with storage at 23°C but was prevented by immediate freezing at ≤ -20°C, without boric acid preservative and without the need to centrifuge urine prior to freezing. CONCLUSIONS: Urinary ATP may have a role as a research tool but is unconvincing as a surrogate, clinical diagnostic marker.

Myo19 ensures symmetric partitioning of mitochondria and coupling of mitochondrial segregation to cell division.

During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure--a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division.