Systems biology approach for mapping the response of human urothelial cells to infection by Enterococcus faecalis

<p>Abstract</p> <p>Background</p> <p>To better understand the response of urinary epithelial (urothelial) cells to <it>Enterococcus faecalis</it>, a uropathogen that exhibits resistance to multiple antibiotics, a genome-wide scan of gene expression was obtained as a time series from urothelial cells growing as a layered 3-dimensional culture similar to normal urothelium. We herein describe a novel means of analysis that is based on deconvolution of gene variability into technical and biological components.</p> <p>Results</p> <p>Analysis of the expression of 21,521 genes from 30 minutes to 10 hours post infection, showed 9553 genes were expressed 3 standard deviations (SD) above the system zero-point noise in at least 1 time point. The asymmetric distribution of relative variances of the expressed genes was deconvoluted into technical variation (with a 6.5% relative SD) and biological variation components (>3 SD above the mode technical variability). These 1409 hypervariable (HV) genes encapsulated the effect of infection on gene expression. Pathway analysis of the HV genes revealed an orchestrated response to infection in which early events included initiation of immune response, cytoskeletal rearrangement and cell signaling followed at the end by apoptosis and shutting down cell metabolism. The number of poorly annotated genes in the earliest time points suggests heretofore unknown processes likely also are involved.</p> <p>Conclusion</p> <p><it>Enterococcus </it>infection produced an orchestrated response by the host cells involving several pathways and transcription factors that potentially drive these pathways. The early time points potentially identify novel targets for enhancing the host response. These approaches combine rigorous statistical principles with a biological context and are readily applied by biologists.</p

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

Type 1 and F17-like Pili Promote the Establishment of the Uropathogenic E. coli Intestinal Reservoir

Urinary tract infections (UTI) affect over 150 million individuals worldwide every year. These infections are associated with significant morbidity and have a sizeable economic impact, with $5 billion being spent on UTI treatment in the USA annually. Uropathogenic E. coli (UPEC) are responsible for 80% of community acquired UTIs and 65% of nosocomial UTI. The current standard of care for UTI is antibiotic therapy. However, 30-50% of women experience recurrent UTI (rUTI) despite receiving antibiotic therapy. The prevalence of single and multi-drug resistant UPEC strains has led to increased reliance on carbepenems, which are primarily reserved for multi-drug resistant infections, to treat an increasing number of patients. Consequently E. coli strains resistant to colistin, a drug of last resort for treating carbapenem-resistant Enterobacteriaceae, have now been isolated in over 30 counties. The rate at which drug-resistant E. coli are spreading across the globe emphasizes the urgent nature of the antibiotic-resistance crisis we currently face and the dire need for new, antibiotic-sparing therapies that target UPEC virulence factors. To colonize host tissue, E. coli encode chaperone usher pathway (CUP) pili and single UPEC strains can encode up to 16 distinct CUP pilus operons. Although CUP pilus types that promote UPEC colonization of the urinary tract have been identified, no studies investigating a role for CUP pilus types in UPEC intestinal colonization have been published. Still, leading models of infection posit that UPEC in the gut seed UTI by being shed in the feces, and then colonizing the peri-urethral or vaginal tissue and subsequently ascending through the urethra to access the bladder. Using a mouse model of UPEC intestinal colonization I found that two (CUP) pili, F17-like pili and type 1 pili, provide a fitness advantages for UPEC in the gut. The X-ray crystal structure of the F17-like pilus adhesin lectin domain, coupled with molecular studies of glycan binding, disclosed a ligand specificity distinct from other pilus types known to facilitate gastrointestinal colonization. While phylogenomic studies revealed that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, I found that high-affinity mannose analogues (mannosides) that target the function of type 1 pili and effectively treat UTI reduce intestinal colonization by clinical UPEC isolates, including a multi-drug resistant strain, without disrupting gut microbiota structure. By decreasing the intestinal UPEC reservoir that seeds bladder infection, mannosides could significantly reduce the rate of UTI and rUTI. Further, mannosides act like a molecular scalpel, specifically targeting intestinal UPEC with minimal effects on the overall gut microbiota. Ultimately, therapies like mannosides, which selectively target colonization by a specific pathogen, have potential to revolutionize treatment and prevention of rUTI. Take together, this dissertation provides invaluable tools and insights into the understudied UPEC intestinal reservoir

Proceedings of the Fourth Annual Conference of the MidSouth Computational Biology and Bioinformatics Society

<p/> <p/> <p/

Aerococcus Urinae: Establishing the Pathogenesis of an Emerging Uropathogen

Urinary tract infection (UTI) is the world\u27s most common bacterial infection. Much is known about the infectious process (pathogenesis) of a few of the bacteria that cause these infections, especially E. coli. Unfortunately, the pathogenesis of E. coli and other uropathogenic bacteria was explored almost exclusively in the belief that the bladder is supposed to be sterile. Our recent evidence, however, debunks this dogma. We used modern methods to reveal diverse bacterial communities in the bladders of adult women. These communities differ in women with and without lower urinary tract symptoms (LUTS), including UTI and urinary incontinence (UI). Many bacteria that we have detected in women with LUTS are understudied precisely because they were previously undetected or overlooked. Thus, very little is known about their pathogenesis. Aerococcus urinae is one of those understudied uropathogenic bacteria. It is associated with both UTI and UI. It is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening sepsis. Thus, I have begun a study of A. urinae\u27s pathogenesis. For well-studied uropathogens, the earliest stages of pathogenesis involve attachment to the cells that line the bladder wall (urothelium) and subsequent disruption of the host\u27s bladder immune system. I hypothesized that A. urinae also attaches to the urothelium and alters signaling to the host\u27s bladder immune system. To test my hypothesis, I first studied in vitro phenotypes of A. urinae related to attachment and colonization of the urothelium. Then, I studied the interaction between human urothelium and A. urinae strains isolated from womenwith LUTS. Results from this dissertation could be used to develop therapies that specifically target A. urinae

Missed urinary tract infection in patients with chronic recalcitrant LUTS and recurrent cystitis

Background: MSU culture and Urinary dipsticks as a diagnostic method for urinary infection (UTI) are discredited despite commonly used to exclude UTI in patients with lower urinary tract symptoms (LUTS). The phenotype of painful LUTS has been recast as Interstitial Cystitis (IC) or Bladder Pains Syndrome (BPS) because infection has been excluded on the evidence of these methods. Given that these all-important tests have been found insensitive and misleading, there is justification in re-examining IC/BPS to ascertain whether we have been mistaken. I studied patients with “Chronic recalcitrant bladder pain and recurrent cystitis” (abbreviated “painful LUTS”) who had been diagnosed with IC/PBS in order to re-assess their pathophysiology.// Aim: I characterised these patients using the scientific method of consilience, which scrutinised them from unrelated perspectives. These studies implied that infection was a most probable aetiological factor. Therefore, I moved on to test infection as a causal factor using Pearl’s three rungs of causation: Correlation, intervention and the counterfactual.// Methods: Data on quality of life and disease experience were obtained. Symptoms and pathophysiological variables in 146 patients presenting with painful LUTS were studied. To achieve Pearl’s specifications, an observational study studied intervention and a cross-over study analysed the counter factual of arbitrary treatment cessation. The evolution of treatment of these patients, using first generation, narrow spectrum urinary agents in protracted courses is reported. Since protracted antibiotic exposure is feared as a cause of antimicrobial resistance (AMR), I measured this in order to round off my findings// Results: The consilience studies incriminated UTI in the aetiology of painful LUTS. It is also clear that the patients suffer terribly, and this is aggravated by professional scepticism catalysed by a misinterpretation of urinalysis data. Antibiotic intervention demonstrated a regression in all disease indicators but there was resurgence of symptoms and signs during trials without treatment. The data on AMR demonstrated a rise in resistance in response to a first prescription without this increasing with persistence of the antibiotic regimen.// Conclusion: These data imply that IC/BPS (painful LUTS) is caused by a treatable urinary tract infection and are sufficient to merit a RCT. Whilst, treatment requires protracted exposure to antibiotics, my data on AMR amongst these patients is surprisingly reassuring. This requires further exploration. Contemporaneous to this thesis, other have published definitive data that refute urine culture and dipstick analysis./

Role of bacterial cystitis in female refractory detrusor overactivity

Background: The aetiology of refractory urge incontinence (also known as refractory Detrusor Overactivity, rDO) remains unknown. However, about 40% of patients experience recurrent bacterial cystitis. The underlying pathophysiology, and the link between inflammation and urgency, haven't been elucidated. The therapeutic implications have not been explored. Methods: Five studies were undertaken. The first study characterised the presence of intracellular bacteria in women with DO. Then a longitudinal study of fluctuations in the urinary microbiome of rDO women with recurrent UTI was performed, using both 16sRNA PCR and traditional culture. The therapeutic effect of 6 weeks of rotating antibiotics in rDO was undertaken, by randomised placebo controlled trial (RCT). Finally, 2 studies explored cytokine levels in rDO women. Because inflammatory cytokines may stimulate afferent nerves to promote urgency, 9 of these chemical messengers were measured in the urine of all RCT enrolees at all visits. Lastly, a much larger study of 27 cytokines (multiplex array) was conducted in a separate cohort of rDO (+/-UTI) vs control. Results: Bacterial filaments were significantly greater in DO women vs controls (82%vs27% p<0.0001, n=88), and intracelluar bacteria were confirmed by confocal microscopy. Over time, the urinary microbiome of rDO women (n=39) was highly diverse and fluctuated (median 25 bacterial genera per urine). “Mixed growth” cultures frequently revealed clear evidence of uropathogens on PCR. The RCT (n=36) showed significant reduction in urinary leakage on 24hr pad test (-75g at 6months) on active treatment. However, 42% of placebo patients needed “clinical override” antibiotics for UTIs. In the RCT, bacteriuria was associated with higher urinary cytokines (in 5 of 9: IL1 α, IL1ra, IL6, IL8, CXCL10), which declined after antibiotics, long term. The large longitudinal study (n=139) of 27 cytokines revealed a striking elevation of both pro and anti-inflammatory cytokines in rDO vs control. Conclusion: Initial data supported the hypothesis that bacterial cystitis promotes refractory urgency. In the RCT, antibiotics suppressed cytokines, which was associated with improved urge incontinence. Overall, the cystitis induced prolonged inflammation appeared to promote cytokine release, allowing enhanced afferent nerve stimuli with resulting refractory urgency. These novel insights open the way for new therapeutic options

Sub-Inhibitory Antibiotics Enhance Virulence, Persistence, and Pathogenesis of Uropathogens

In addition to their bactericidal effects, antibiotics are potent signal mediators at sub-inhibitory levels in the environment. The ability to modulate community structure in this niche raises concerns over their capacity to influence pathogenesis in patients during antibiotic therapy. This concept forms the basis of this thesis, and is explored using models of prophylactic therapy for recurrent urinary tract infection (UTI) management. Sub-inhibitory ciprofloxacin, ampicillin, and gentamicin were found to augment virulence in vitro, increasing adherence and urothelial cell invasion in uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus. In addition, biofilm formation was increased, and swarming motility decreased. In UPEC, the effect of antibiotics on these processes was abolished in SOS-deficient strains. Trans-urethral inoculation of mice with ciprofloxacin-primed S. saprophyticus or UPEC significantly increased bacterial burden in both bladders and kidneys at one and 14 days post-inoculation (dpi). Sub-therapeutic ciprofloxacin supplemented in the drinking water of chronically infected mice significantly increased bacterial urine load. In addition, mice previously infected but clinically resolved suffered recurrences. These mice had impaired urinary polymorphonuclear leukocyte infiltrates, in part due to antibiotic-dependent cytokine suppression during initial infection. Prophylactic intervention had no significant effect on UPEC clearance, but did significantly increase bacterial intracellular bladder reservoirs, raising concerns over the clinical efficacy of this management strategy and risks of promoting persistent infection. The inability of antibiotics to clear infection in prophylaxis models was attributed to the presence of MDT persister cells. Sub-inhibitory antibiotic pre-treatments were found to increase persister fractions, but this effect was abolished in SOS-deficient strains. Conducting these assays with UPEC isolated from recurrent UTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy, suggesting persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure. This work represents the first attempt to illustrate that observed sub-inhibitory, pathogen associated antibiotic-dependent changes in vitro have significant in vivo consequences. It is hoped that this research will lead to a re-examination of how antibiotics are administered for management of patients suffering from recurrent UTI and other chronic diseases

Raman spectroscopy for point of care urinary tract infection diagnosis

Urinary tract infections (UTIs) are one of the most common bacterial infections experience by humans, with 150 million people suffering one or more UTIs each year. The massive scale at which UTIs occurs translates to a tremendous health burden comprising of patient morbidity and mortality, massive societal costs and a recognised contribution to expanding antimicrobial resistance. The considerable disease burden caused by UTIs is severely exacerbated by an outdated diagnostic paradigm characterised by inaccuracy and delay. Poor accuracy of screening tests, such as urinalysis, lead to misdiagnosis which in turn result in delayed recognition or overtreatment. Additionally, these screening tests fail to identify the causative pathogen, causing an overreliance on broad-spectrum antimicrobials which exacerbate burgeoning antimicrobial resistance. While diagnosis may be accurately confirmed though culture and sensitivity testing, the prolonged delay incurred negates the value of the information provided doing so. A novel diagnostic paradigm is required that that targets rapid and accurate diagnosis of UTIs, while providing real-time identification of the causative pathogen. Achieving this precision management is contingent on the development of novel diagnostic technologies that bring accurate diagnosis and pathogen classification to the point of care. The purpose of this thesis is to develop a technology that may form the core of a point-of-care diagnostic capable of delivering rapid and accurate pathogen identification direct from urine sample. Raman spectroscopy is identified as a technology with the potential to fulfil this role, primarily mediated though its ability to provide rapid biochemical phenotyping without requiring prior biomass expansion. Raman spectroscopy has demonstrated an ability to achieve pathogen classification through the analysis of inelastically scattered light arising from pathogens. The central challenge to developing a Raman-based diagnostic for UTIs is enhancing the weak bacterial Raman signal while limiting the substantial background noise. Developing a technology using Raman spectroscopy able to provide UTI diagnosis with uropathogen classification is contingent on developing a robust experimental methodology that harnesses the multitude of experimental and analytical parameters. The refined methodology is applied in a series of experimental works that demonstrate the unique Raman spectra of pathogens has the potential for accurate classification. Achieving this at a clinically relevant pathogen load and in a clinically relevant timeframe is, however, dependent on overcoming weak bacterial signal to improve signal-to-noise ratio. Surface-enhanced Raman spectroscopy (SERS) provides massive Raman signal enhancement of pathogens held in close apposition to noble metal nanostructures. Additionally, vacuum filtration is identified as a means of rapidly capturing pathogens directly from urine. SERS-active filters are developed by applying a gold nanolayer to commercially available membrane filters through physical vapour deposition. These SERS-active membrane filter perform multiple roles of capturing pathogens, separating them from urine, while providing Raman signal enhancement through SERS. The diagnostic and classification performance of SERS-active filters for UTIs is demonstrated to achieve rapid and accurate diagnosis of infected samples, with real-time uropathogen classification, using phantom urine samples, before piloting the technology using clinical urine samples. The Raman technology developed in this thesis will be further developed toward a clinically implementable technology capable of ameliorating the substantial burden of disease caused by UTIs.Open Acces