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.

Management of patients who opt for radical prostatectomy during the coronavirus disease 2019 (COVID-19) pandemic: an international accelerated consensus statement.

OBJECTIVE: Coronavirus disease-19 (COVID-19) pandemic caused delays in definitive treatment of patients with prostate cancer. Beyond the immediate delay a backlog for future patients is expected. The objective of this work is to develop guidance on criteria for prioritisation of surgery and reconfiguring management pathways for patients with non-metastatic prostate cancer who opt for surgical treatment. A second aim was to identify the infection prevention and control (IPC) measures to achieve a low likelihood of coronavirus disease 2019 (COVID-19) hazard if radical prostatectomy (RP) was to be carried out during the outbreak and whilst the disease is endemic. METHODS: We conducted an accelerated consensus process and systematic review of the evidence on COVID-19 and reviewed international guidance on prostate cancer. These were presented to an international prostate cancer expert panel (n = 34) through an online meeting. The consensus process underwent three rounds of survey in total. Additions to the second- and third-round surveys were formulated based on the answers and comments from the previous rounds. The Consensus opinion was defined as ≥80% agreement and this was used to reconfigure the prostate cancer pathways. RESULTS: Evidence on the delayed management of patients with prostate cancer is scarce. There was 100% agreement that prostate cancer pathways should be reconfigured and measures developed to prevent nosocomial COVID-19 for patients treated surgically. Consensus was reached on prioritisation criteria of patients for surgery and management pathways for those who have delayed treatment. IPC measures to achieve a low likelihood of nosocomial COVID-19 were coined as 'COVID-19 cold' sites. CONCLUSION: Reconfiguring management pathways for patients with prostate cancer is recommended if significant delay (>3-6 months) in surgical management is unavoidable. The mapped pathways provide guidance for such patients. The IPC processes proposed provide a framework for providing RP within an environment with low COVID-19 risk during the outbreak or when the disease remains endemic. The broader concepts could be adapted to other indications beyond prostate cancer surgery.

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.

A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination.

INTRODUCTION AND HYPOTHESIS: Midstream urine (MSU) is key in assessing lower urinary tract syndrome (LUTS), but contingent on some assumptions. The aim of this study was to compare the occurrence of contamination and the quality of substrates obtained from four different collections: MSU, catheter specimen urine (CSU), a commercial MSU collecting device (Peezy) and a natural void. Contamination was quantified by differential, uroplakin-positive, urothelial cell counts. METHODS: This was a single blind, crossover study conducted in two phases. First, we compared the MSU with CSU using urine culture, pyuria counts and differential counting of epithelial cells after immunofluorescence staining for uroplakin III (UP3). Second, we compared the three non-invasive (MSU, Peezy MSU™, natural void) methods using UP3 antibody staining only. RESULTS: The natural void was best at collecting bladder urinary sediment, with the majority of epithelial cells present derived from the urinary tract. CSU sampling missed much of the urinary sediment and showed sparse culture results. Finally, the MSU collection methods did not capture much of the bladder sediment. CONCLUSION: We found little evidence for contamination with the four methods. Natural void was the best method for harvesting shed urothelial cells and white blood cells. It provides a richer sample of the inflammatory exudate, including parasitised urothelial cells and the microbial substrate. However, if the midstream sample is believed to be important, the MSU collection device is advantageous.

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).

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.

Differential regulation of actin microfilaments by human MICAL proteins.

The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation.

Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms.

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

Discrediting microscopic pyuria and leucocyte esterase as diagnostic surrogates for infection in patients with lower urinary tract symptoms: results from a clinical and laboratory evaluation.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Microscopic pyuria is widely used as a surrogate marker of infection, although there is little data supporting its use in patients who present with non-acute LUTS. The effects of urinary storage, preservation, and the use of laboratory methods to enhance leucocyte detection, are also unclear. This large, prospective study highlights the poor performance of dipstick urine analysis, and direct microscopy, as surrogate markers of UTI in patients with LUTS. A series of laboratory analyses also examine the effects of urine handling and processing on test integrity, which have important implications for clinical practice. OBJECTIVE: To evaluate the diagnostic performance of pyuria as a surrogate marker of urinary tract infection (UTI) in patients with chronic lower urinary tract symptoms (LUTS), and determine the impact of sample storage, cytocentrifugation, and staining techniques, on test performance. PATIENTS AND METHODS: Between 2008 and 2011, we recruited 1223 patients (120 men; 1103 women; mean age 54 years) with one or more LUTS from a specialist urological outpatient service. We conducted a prospective observational study to determine the performance of microscopic pyuria ≥10 wbc/µL as a surrogate marker of UTI in patients with LUTS. All patients provided clean-catch midstream urine (MSU) samples for analysis, and routine microbiological cultures were used as our reference standard. We also scrutinised the performance of dipstick leucocyte esterase ≥ 'trace' in the detection of microscopic pyuria. The influence of sample handling and processing on test performance was examined in a series of laboratory studies. The effects of storage on leucocyte decay were determined using repeated microscopic assessments of individual urine samples, to plot temporal changes in leucocyte numbers. This study used varied storage conditions (≈20 °C and 4 °C), and boric acid preservation. Paired microscopic assessments were used to determine the effects of centrifugation on leucocyte salvage in spun/unspun samples (relative centrifugal force range 39-157 g). Similar methods were used to assess microscopic leucocyte quantification in stained/unstained urine (Sternheimer-Malbin protocol). RESULTS: The positive predictive value (PPV) and negative predictive value (NPV) of pyuria as a surrogate marker of UTI were 0.40 (95% confidence interval [CI] 0.37-0.43) and 0.75 (95% CI 0.73-0.76), respectively. The dipstick was unable to identify significant microscopic pyuria (≥10 wbc/µL) in 60% of the samples: PPV 0.51 (95% CI 0.48-0.55); NPV 0.75 (95% CI 0.73-0.76). Microscopic pyuria performed poorly as a surrogate of UTI defined by bacterial culture. Whilst refrigeration and preservation did retard leucocyte loss (F = 11; DF = 2; P < 0.001), 40% of cells were still lost by 4 h. Centrifugation had an unpredictable influence on cell salvage (coefficient of variation 5750%) and the use of staining to improve leucocyte detection proved ineffective (Z = -0.356; P = 0.72). CONCLUSIONS: Pyuria performs badly as a surrogate of UTI in patients with LUTS. This is exacerbated by cell loss during storage, and neither centrifugation, nor staining, appears to confer any diagnostic advantage. Clinicians should be alerted to the significant limitations of these tests.

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.

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.

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.

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.

Tao-1 is a negative regulator of microtubule plus-end growth.

Microtubule dynamics are dominated by events at microtubule plus ends as they switch between discrete phases of growth and shrinkage. Through their ability to generate force and direct polar cell transport, microtubules help to organise global cell shape and polarity. Conversely, because plus-end binding proteins render the dynamic instability of individual microtubules sensitive to the local intracellular environment, cyto-architecture also affects the overall distribution of microtubules. Despite the importance of plus-end regulation for understanding microtubule cytoskeletal organisation and dynamics, little is known about the signalling mechanisms that trigger changes in their behaviour in space and time. Here, we identify a microtubule-associated kinase, Drosophila Tao-1, as an important regulator of microtubule stability, plus-end dynamics and cell shape. Active Tao-1 kinase leads to the destabilisation of microtubules. Conversely, when Tao-1 function is compromised, rates of cortical-induced microtubule catastrophe are reduced and microtubules contacting the actin cortex continue to elongate, leading to the formation of long microtubule-based protrusions. These data reveal a role for Tao-1 in controlling the dynamic interplay between microtubule plus ends and the actin cortex in the regulation of cell form.