Purification of Intracellular Bacterial Communities during Experimental Urinary Tract Infection Reveals an Abundant and Viable Bacterial Reservoir

ABSTRACTUrinary tract infections (UTIs) are a major infection of humans, particularly affecting women. Recurrent UTIs can cause significant discomfort and expose patients to high levels of antibiotic use, which in turn contributes to the development of higher antibiotic resistance rates. Most UTIs are caused by uropathogenicEscherichia coli, which is able to form intracellular collections (termed intracellular bacterial communities [IBCs]) within the epithelial cells lining the bladder lumen. IBCs are seen in both infected mice and humans and are a potential cause of recurrent UTI. Genetic and molecular studies of IBCs have been hampered both by the low number of bacteria in IBCs relative to the number extracellular bacteria and by population bottlenecks that occur during IBC formation. We now report the development of a simple and rapid technique for isolating pure IBCs from experimentally infected mice. We verified the specificity and purity of the isolated IBCs via microscopy, gene expression, and culture-based methods. Our results further demonstrated that our isolation technique practically enables specific molecular studies of IBCs. In the first such direct measurement, we determined that a single epithelial cell containing an early IBC typically contains 103viable bacteria. Our isolation technique complements recent progress in low-input, single-cell genomics to enable future genomic studies of the formation of IBCs and their activation pathways during recurrent UTI, which may lead to novel strategies to eliminate them from the bladder.

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Assessment and treatment of recurrent urinary tract infections in women: development of a questionnaire based on a qualitative study of patient expectations in secondary care

BMC Urology ◽

10.1186/s12894-020-00764-6 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

J. J. Pat ◽

T. v. d. Aart ◽

M. G. Steffens ◽

L. P. W. Witte ◽

M. H. Blanker

Keyword(s):

Urinary Tract ◽

Secondary Care ◽

Prophylactic Antibiotic ◽

Antibiotic Use ◽

Patient Expectations ◽

Structured Interviews ◽

Daily Lives ◽

Recurrent Uti ◽

Assessment And Treatment ◽

Tract Infections

Abstract Background To develop a questionnaire to facilitate the inventorying of women’s expectations for the assessment and treatment of recurrent urinary tract infection (UTI) in secondary care. Methods Semi-structured interviews were conducted among women with recurrent UTI referred to our urology department. The interviews were conducted by one interviewer, recorded, transcribed verbatim, and analyzed thematically by two researchers. We first developed 35 questions to identify potential themes, and we then tested them among women with and without recurrent UTI. Changes were made according to the feedback received. Results Six interviews were conducted before saturation was reached. Thematic analysis identified three themes: patient pathway, personal knowledge, and social implications. All respondents had received multiple antibiotic courses but no prophylactic antibiotic therapy, and although all were aware of some preventive measures, they wanted more information about their disease. However, some women were afraid to access information for fear of what they might learn. Recurrent UTI also significantly affected the daily lives all respondents. Some women expressed fears over frequent antibiotic use, and others felt that there must be something wrong with their body to have so many UTIs. Women expected the urologist to provide an explanation and to start adequate therapy for their recurrent UTI. We created a 32-item questionnaire based on these themes Conclusion This study not only developed a questionnaire for use when assessing patient expectations of recurrent UTI management in secondary care but also provided novel insights into the thoughts, opinions, and expectations of women who are referred.

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Involvement of biofilm-like in tracellular bacterial communities of uropathogenic Escherichia coli in pathogenesis of urinary tract infections – a mini review

Current Issues in Pharmacy and Medical Sciences ◽

10.12923/j.2084-980x/26.3/a.18 ◽

2013 ◽

Vol 26 (3) ◽

pp. 321-325

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Bacterial Communities ◽

Uropathogenic Escherichia Coli ◽

E Coli ◽

Etiologic Agents ◽

Tract Infections ◽

Intracellular Bacterial Communities

This paper presents a precisely defined question about the role of the biofilm-like intracellular bacterial communities in pathogenesis of the urinary tract infections. According to the recent literature, uropathogenic Escherichia coli is one of the leading etiologic agents of the urinary tract infections. Although E. coli is regarded as an extracellular pathogen, some experiments have revealed a multi-step infection cycle, which involves adhesion, invasion, proliferation within invaded urothelial cell in the form of biofilm-like intracellular bacterial communities and dispersal, leading to infection of next neighbouring cells. Therefore, the prevention and treatment of the urinary tract infections must include intracellular stage of infection.

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Metabolic Requirements ofEscherichia coliin Intracellular Bacterial Communities during Urinary Tract Infection Pathogenesis

mBio ◽

10.1128/mbio.00104-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 40

Author(s):

Matt S. Conover ◽

Maria Hadjifrangiskou ◽

Joseph J. Palermo ◽

Michael E. Hibbing ◽

Karen W. Dodson ◽

...

Keyword(s):

Oxidative Stress ◽

Urinary Tract ◽

Bacterial Communities ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Bladder Cells ◽

Tract Infections ◽

Intracellular Bacterial Communities

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly,lacZwas highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZstrain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, agalKmutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated geneyeaRrevealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies.IMPORTANCEUrinary tract infections (UTIs) are one of the most common bacterial infections, impacting mostly women. Every year, millions of UTIs occur in the U.S. with most being caused by uropathogenicE. coli(UPEC). During a UTI, UPEC invade bladder cells and form an intracellular bacterial community (IBC) that allows for the bacteria to replicate protected from the host immune response. In this study, we investigated genes that are expressed by UPEC within the IBC and determined how they contribute to the formation of this specialized community. Our findings suggest that galactose is important for UPEC growth in the IBC. Additionally, we found that a gene involved in oxidative stress is also important in the regulation of a key factor needed for UPEC invasion of bladder cells. These results may open the door for the development of treatments to diminish UTI frequency and/or severity.

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Draft Genome Sequence of Klebsiella pneumoniae UMB7779, Isolated from the Female Urinary Tract

Microbiology Resource Announcements ◽

10.1128/mra.00396-20 ◽

2020 ◽

Vol 9 (20) ◽

Author(s):

Pavan Bhimalli ◽

Taylor Miller-Ensminger ◽

Adelina Voukadinova ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Urinary Tract Infections ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Content Type ◽

Recurrent Uti ◽

Tract Infections ◽

Female Urinary Tract

Klebsiella pneumoniae is a pathogenic bacterium commonly responsible for urinary tract infections (UTIs). Here, we report the draft genome sequence of K. pneumoniae strain UMB7779, isolated from catheterized urine of a woman with a recurrent UTI.

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Abnormal coagulation studies associated with levoflaxacin

Journal of Neurosurgery ◽

10.3171/jns.2004.100.4.0710 ◽

2004 ◽

Vol 100 (4) ◽

pp. 710-712 ◽

Cited By ~ 4

Author(s):

Thomas Psarros ◽

Talmadge Trammell ◽

Kevin Morrill ◽

Cole Giller ◽

Howard Morgan ◽

...

Keyword(s):

Urinary Tract ◽

Antibiotic Use ◽

Acquired Von Willebrand Syndrome ◽

Content Type ◽

Coagulation Abnormalities ◽

Neurosurgical Patients ◽

Tract Infections ◽

Spine Disorders ◽

Von Willebrand ◽

Fine Print

✓ Complications arising from antibiotic use are of interest to neurosurgeons because many neurosurgical patients are treated for infection. In this report, the authors describe three patients with spine disorders who developed coagulopathies after treatment with levofloxacin, an antibiotic commonly used by neurosurgical services. Three patients with spine disorders developed urinary tract infections (UTIs) for which they received a 3-day course of oral levofloxacin. Subsequently, they demonstrated prolonged prothrombin times and increased international normalized ratios. One of those patients later developed acquired von Willebrand syndrome during surgery. Coagulopathies were successfully corrected preoperatively with parenteral vitamin K. The patient with acquired von Willebrand syndrome required multiple transfusions. There seems to be an association between levofloxacin and coagulation abnormalities in neurosurgical patients treated for UTIs. Neurosurgical services prescribing this common antibiotic should be aware of this problem.

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Uropathogenic E. coli induces DNA damage in the bladder

10.1101/2020.05.07.080291 ◽

2020 ◽

Author(s):

Camille V. Chagneau ◽

Clémence Massip ◽

Nadège Bossuet-Greif ◽

Christophe Fremez ◽

Jean-Paul Motta ◽

...

Keyword(s):

Colorectal Cancer ◽

Escherichia Coli ◽

Dna Damage ◽

Bladder Cancer ◽

Urinary Tract ◽

Bacterial Communities ◽

E Coli ◽

Bladder Cells ◽

Tract Infections ◽

Intracellular Bacterial Communities

AbstractUrinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of a metabolite released during the synthesis of colibactin, a bacterial genotoxin, in 50 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells. This bacterial genotoxin, which is increasingly suspected to promote colorectal cancer, should also be scrutinised in the context of bladder cancer.

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Assessment and treatment of recurrent urinary tract infections in women: qualitative study of patient expectations in secondary care

10.21203/rs.3.rs-51682/v2 ◽

2020 ◽

Author(s):

Jorik Pat ◽

Tamar van der Aart ◽

Martijn Steffens ◽

Lambertus Witte ◽

Marco Blanker

Keyword(s):

Urinary Tract ◽

Secondary Care ◽

Prophylactic Antibiotic ◽

Antibiotic Use ◽

Patient Expectations ◽

Structured Interviews ◽

Daily Lives ◽

Recurrent Uti ◽

Assessment And Treatment ◽

Tract Infections

Abstract Background. To develop a questionnaire to facilitate the inventorying of women’s expectations for the assessment and treatment of recurrent urinary tract infection (UTI) in secondary care.Methods. Semi-structured interviews were conducted among women with recurrent UTI referred to our urology department. The interviews were conducted by one interviewer, recorded, transcribed verbatim, and analyzed thematically by two researchers. We first developed 35 questions to identify potential themes, and we then tested them among women with and without recurrent UTI. Changes were made according to the feedback received.Results. Six interviews were conducted before saturation was reached. Thematic analysis identified three themes: patient pathway, personal knowledge, and social implications. All respondents had received multiple antibiotic courses but no prophylactic antibiotic therapy, and although all were aware of some preventive measures, they wanted more information about their disease. However, some women were afraid to access information for fear of what they might learn. Recurrent UTI also significantly affected the daily lives all respondents. Some women expressed fears over frequent antibiotic use, and others felt that there must be something wrong with their body to have so many UTIs. Women expected the urologist to provide an explanation and to start adequate therapy for their recurrent UTI. We created a 32-item questionnaire based on these themesConclusion. This study not only developed a questionnaire for use when assessing patient expectations of recurrent UTI management in secondary care but also provided novel insights into the thoughts, opinions, and expectations of women who are referred.

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Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections

10.1101/2021.01.03.424836 ◽

2021 ◽

Author(s):

Kunal Sharma ◽

Neeraj Dhar ◽

Vivek V. Thacker ◽

Thomas Simonet ◽

François Signorino-Gelo ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Epithelial Cells ◽

Urinary Tract Infections ◽

Bacterial Communities ◽

Antibiotic Administration ◽

Human Bladder ◽

Tract Infections ◽

Intracellular Bacterial Communities

AbstractUropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections and is a major cause for antibiotic prescriptions. Previous studies have shown that infection of terminally differentiated urinary bladder cells leads to the formation of intracellular bacterial communities (IBCs). However, the precise role of IBCs in recurrence of infection and antibiotic persistence, is not completely understood in part because the in situ dynamic responses of bacteria within these structures to antibiotic stress is difficult to assess in animal models. Here, we develop and characterize a human bladder-chip model of UPEC infection wherein superficial bladder epithelial cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and the mechanics of bladder filling and voiding cycles mimicked by application and release of linear strain. Time-lapse microscopy showed that infection of epithelial cells under shear stress in diluted urine led to the rapid recruitment and diapedesis of neutrophils across the endothelial-epithelial barrier and the formation of neutrophil swarms and neutrophil extracellular traps. Subsequently, two cycles of antibiotic administration interspersed with recovery periods revealed both non-growing and rapidly proliferating IBCs. Multiple stages of IBC formation captured on-chip with single-cell resolution revealed that that bacterial killing within IBCs was substantially delayed, outcomes such as shedding of bacteria and exfoliation are not mutually exclusive and rapidly reseeded the infection, and in rare instances bacterial growth in IBCs continued for the entire period of antibiotic administration. These new insights into the early stages of pathogenesis revisit the role of IBCs as harbours of persistent bacterial populations, with significant consequences for non-compliance with antibiotic regimens.

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Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection

eLife ◽

10.7554/elife.66481 ◽

2021 ◽

Vol 10 ◽

Author(s):

Kunal Sharma ◽

Neeraj Dhar ◽

Vivek V Thacker ◽

Thomas M Simonet ◽

Francois Signorino-Gelo ◽

...

Keyword(s):

Urinary Tract ◽

Bacterial Communities ◽

Recovery Period ◽

Dynamic Responses ◽

Neutrophil Extracellular Trap ◽

Antibiotic Administration ◽

Human Bladder ◽

Tract Infections ◽

Intracellular Bacterial Communities ◽

Umbrella Cells

Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

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Uropathogenic E. coli induces DNA damage in the bladder

PLoS Pathogens ◽

10.1371/journal.ppat.1009310 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009310

Author(s):

Camille V. Chagneau ◽

Clémence Massip ◽

Nadège Bossuet-Greif ◽

Christophe Fremez ◽

Jean-Paul Motta ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Bacterial Communities ◽

Cleavage Product ◽

E Coli ◽

Bladder Cells ◽

Tract Infections ◽

Intracellular Bacterial Communities

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

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