The Urinary Microbiome: Improving Diagnostics and Management of Urinary Tract Infections in Adult Females.

This article discusses the urinary microbiome in relation to urinary tract infection (UTI) in women. It makes biologic sense that the microbiota of different niches (bladder, vagina, and gut) interact with each other in health, as well as during a UTI event; however, these relationships remain poorly understood. Future research should close knowledge gaps regarding the interactions between the urinary microbiota and the host, amongst the microbiota of adjacent niches, and between the microbes within the same microbiota. The new knowledge should result in improved UTI treatment in the age of antibiotic stewardship.

Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

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

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

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

Comprehensive analysis of multiple cytokines to stratify uropathogenic Escherichia coli pathogenesis in mouse model of urinary tract infection.

PURPOSE: Urinary tract infection (UTI) is one of the most common human bacterial infections primarily caused by uropathogenic E. coli (UPEC). Empiric treatment in UTI cause emergence of multidrug resistance and limit treatment options. Understanding UTI at the molecular level with respect to the causative pathogen as well as subsequent host response pose an absolute necessity towards appropriate clinical management. This study aimed to investigate host cytokine response in mouse UTI model with respect to bacterial colonization and associated virulence gene expression upon infection. METHOD: Mouse UTI model was established with two clinical UPEC isolates E. coli NP105 and E. coli P025. UPEC colonization in bladder and kidney was evaluated by bacterial culture (CFU/ml). Histopathology of the tissues were examined by hematoxylin and eosin staining. PCR and real time PCR were used to detect the incidence and expression of respective bacterial genes. Cytokine concentrations in tissues and sera were evaluated using ELISA. GraphPad prism version 8.0.2 was used for statistical interpretation. RESULT: Highest bacterial colonization was observed on 7th and 9th day post infection (p.i). in bladder and kidney of mouse infected with E. coli P025 and E. coli NP105 respectively with a distinct difference in relative expression of fimH and papC adhesin genes in vivo. IL-1ß level in tissues and sera of E. coli NP105 and E. coli P025 infected mouse was significantly different but the IL-17A, GCSF, TGF-ß levels were comparable. CONCLUSION: These findings show a role of IL1ß to stratify pathogenicity of UPEC in mouse UTI model.

Cataloging the phylogenetic diversity of human bladder bacterial isolates.

BACKGROUND: Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. RESULTS: To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. CONCLUSIONS: This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.

Insulin receptor signaling engages bladder urothelial defenses that limit urinary tract infection.

Urinary tract infections (UTIs) commonly afflict people with diabetes. To better understand the mechanisms that predispose diabetics to UTIs, we employ diabetic mouse models and altered insulin signaling to show that insulin receptor (IR) shapes UTI defenses. Our findings are validated in human biosamples. We report that diabetic mice have suppressed IR expression and are more susceptible to UTIs caused by uropathogenic Escherichia coli (UPEC). Systemic IR inhibition increases UPEC susceptibility, while IR activation reduces UTIs. Localized IR deletion in bladder urothelium promotes UTI by increasing barrier permeability and suppressing antimicrobial peptides. Mechanistically, IR deletion reduces nuclear factor κB (NF-κB)-dependent programming that co-regulates urothelial tight junction integrity and antimicrobial peptides. Exfoliated urothelial cells or urine samples from diabetic youths show suppressed expression of IR, barrier genes, and antimicrobial peptides. These observations demonstrate that urothelial insulin signaling has a role in UTI prevention and link IR to urothelial barrier maintenance and antimicrobial peptide expression.

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

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

Mapping niche-specific two-component system requirements in uropathogenic Escherichia coli.

Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify-for the first time-a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate-at a systems level-which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

VesiX cetylpyridinium chloride is rapidly bactericidal and reduces uropathogenic Escherichia coli bladder epithelial cell invasion in vitro.

Management of urinary tract infection (UTI) in postmenopausal women can be challenging. The recent rise in resistance to most of the available oral antibiotic options together with high recurrence rate in postmenopausal women has further complicated treatment of UTI. As such, intravesical instillations of antibiotics like gentamicin are being investigated as an alternative to oral antibiotic therapies. This study evaluates the efficacy of the candidate intravesical therapeutic VesiX, a solution containing the cationic detergent Cetylpyridinium chloride, against a broad range of uropathogenic bacterial species clinically isolated from postmenopausal women with recurrent UTI (rUTI). We also evaluate the cytotoxicity of VesiX against cultured bladder epithelial cells and find that low concentrations of 0.0063% and 0.0125% provide significant bactericidal effect toward diverse bacterial species including uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis while minimizing cytotoxic effects against cultured 5637 bladder epithelial cells. Lastly, to begin to evaluate the potential utility of using VesiX in combination therapy with existing intravesical therapies for rUTI, we investigate the combined effects of VesiX and the intravesical antibiotic gentamicin. We find that VesiX and gentamicin are not antagonistic and are able to reduce levels of intracellular UPEC in cultured bladder epithelial cells. IMPORTANCE: When urinary tract infections (UTIs), which affect over 50% of women, become resistant to available antibiotic therapies dangerous complications like kidney infection and lethal sepsis can occur. New therapeutic paradigms are needed to expand our arsenal against these difficult to manage infections. Our study investigates VesiX, a Cetylpyridinium chloride (CPC)-based therapeutic, as a candidate broad-spectrum antimicrobial agent for use in bladder instillation therapy for antibiotic-resistant UTI. CPC is a cationic surfactant that is FDA-approved for use in mouthwashes and is used as a food additive but has not been extensively evaluated as a UTI therapeutic. Our study is the first to investigate its rapid bactericidal kinetics against diverse uropathogenic bacterial species isolated from postmenopausal women with recurrent UTI and host cytotoxicity. We also report that together with the FDA-approved bladder-instillation agent gentamicin, VesiX was able to significantly reduce intracellular populations of uropathogenic bacteria in cultured bladder epithelial cells.

D-Mannose reduces cellular senescence and NLRP3/GasderminD/IL-1ß-driven pyroptotic uroepithelial cell shedding in the murine bladder.

Aging is a risk factor for disease via increased susceptibility to infection, decreased ability to maintain homeostasis, inefficiency in combating stress, and decreased regenerative capacity. Multiple diseases, including urinary tract infection (UTI), are more prevalent with age; however, the mechanisms underlying the impact of aging on the urinary tract mucosa and the correlation between aging and disease remain poorly understood. Here, we show that, relative to young (8-12 weeks) mice, the urothelium of aged (18-24 months) female mice accumulates large lysosomes with reduced acid phosphatase activity and decreased overall autophagic flux in the aged urothelium, indicative of compromised cellular homeostasis. Aged bladders also exhibit basal accumulation of reactive oxygen species (ROS) and a dampened redox response, implying heightened oxidative stress. Furthermore, we identify a canonical senescence-associated secretory phenotype (SASP) in the aged urothelium, along with continuous NLRP3-inflammasome- and Gasdermin-D-dependent pyroptotic cell death. Consequently, aged mice chronically exfoliate urothelial cells, further exacerbating age-related urothelial dysfunction. Upon infection with uropathogenic E. coli, aged mice harbor increased bacterial reservoirs and are more prone to spontaneous recurrent UTI. Finally, we discover that treatment with D-mannose, a natural bioactive monosaccharide, rescues autophagy flux, reverses the SASP, and mitigates ROS and NLRP3/Gasdermin/interleukin (IL)-1ß-driven pyroptotic epithelial cell shedding in aged mice. Collectively, our results demonstrate that normal aging affects bladder physiology, with aging alone increasing baseline cellular stress and susceptibility to infection, and suggest that mannose supplementation could serve as a senotherapeutic to counter age-associated urothelial dysfunction.

Survey of the infant male urobiome and genomic analysis of Actinotignum spp.

The urinary bladder harbors a community of microbes termed the urobiome, which remains understudied. In this study, we present the urobiome of healthy infant males from samples collected by transurethral catheterization. Using a combination of enhanced culture and amplicon sequencing, we identify several common bacterial genera that can be further investigated for their effects on urinary health across the lifespan. Many genera were shared between all samples suggesting a consistent urobiome composition among this cohort. We note that, for this cohort, early life exposures including mode of birth (vaginal vs. Cesarean section), or prior antibiotic exposure did not influence urobiome composition. In addition, we report the isolation of culturable bacteria from the bladders of these infant males, including Actinotignum spp., a bacterial genus that has been associated with urinary tract infections in older male adults. Herein, we isolate and sequence 9 distinct strains of Actinotignum spp. enhancing the genomic knowledge surrounding this genus and opening avenues for delineating the microbiology of this urobiome constituent. Furthermore, we present a framework for using the combination of culture-dependent and sequencing methodologies for uncovering mechanisms in the urobiome.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.

Bladder-draining lymph nodes support germinal center B cell responses during urinary tract infection in mice.

Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multidrug-resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we directly observed host adaptive immune responses in acute UTI. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with a clinically relevant MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1-/- C57BL/6J mice harbored larger bacterial burdens than wild-type counterparts, consistent with a role for adaptive immunity in UTI control. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11chi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal center B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice and, although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model that UPEC UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.

Ascending renal infection following experimental candiduria by Candida tropicalis in immunocompromised mice.

The present study evaluated renal infection resulting from the implantation of C. tropicalis in the bladder of immunosuppressed mice. Yeasts were implanted in two manners: planktonic and via preformed biofilm on a small catheter fragment (SCF). Renal histopathology and cultures was performed 72 and 144 h after cystotomy was carried out in mice from three groups: group I contained non-contaminated mice implanted with a sterile SCF; group II mice received a sterile SCF plus a yeast suspension containing 1 × 107 yeasts/mL in a planktonic form; group III mice were implanted with a SCF containing preformed C. tropicalis biofilm. Viable yeasts were found in the kidneys of mice from both groups II and III. However, after 72 h the planktonic cells (group II) invaded more quickly than the sessile cells (group III). Over a longer period (144 h), group III exhibited a more invasive infection (50% of the animals presented renal infection and the renal fungal load was 3.2 log10 CFU/g tissue) than in group II, where yeasts were not found. C. tropicalis introduced into the bladder in two ways (in planktonic or biofilm form) were able to reach the kidney and establish a renal fungal infection, causing interstitial disorders. The data of the present study therefore support the hypothesis of an ascending pathway for renal infections by C. tropicalis. Furthermore, the biofilm resulted in a greater and progressive risk of renal infection, attributed to the slow detachment of the yeasts.

Can antibiotics for enteritis or for urinary tract infection disrupt the urinary microbiota in rats?

Background: To establish antibiotic preregimes and administration routes for studies on urinary microbiota. Methods and materials: Antibiotics for enteritis (Abx-enteritis) and UTIs (Abx-UTI) were administered via gavage and/or urinary catheterisation (UC) for 1 and/or 2 weeks. The effects of these Abx on the urinary microbiota of rats were examined via 16S rRNA sequencing and urine culture, including anaerobic and aerobic culture. Additionally, the safety of the Abx was examined. Results: Abx-enteritis/Abx-UTI (0.5 g/L and 1 g/L) administered via gavage did not alter the microbial community and bacterial diversity in the urine of rats (FDR > 0.05); however, Abx-UTI (1 g/L) administered via UC for 1 and 2 weeks altered the urinary microbial community (FDR < 0.05). Rats administered Abx-UTI (1 g/L) via UC for 1 week demonstrated a distinct urinary microbiota in culture. Abx-enteritis/Abx-UTI administered via gavage disrupted the microbial community and reduced bacterial diversity in the faeces of rats (FDR < 0.05), and Abx-UTI administered via UC for 2 weeks (FDR < 0.05) altered the fecal microbiota. Abx-UTI (1 g/L) administered via UC did not alter safety considerations. In addition, we noticed that UC did not induce infections and injuries to the bladder and kidney tissues. Conclusions: Administration of Abx-UTI via UC for 1 week can be considered a pre-treatment option while investigating the urinary microbiota.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

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.

Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.