scholarly journals An Optimized Fluorometric Method to Test the Capacities of Cranberry Polyphenols and Metabolites to Inhibit the Adhesion of Type-P and Type-1 Fimbriated Uropathogenic E. coli

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 593-593
Author(s):  
Shuhan Li ◽  
Liwei Gu ◽  
Shaomin Zhao ◽  
Yavuz Yagiz
Keyword(s):  
Urinary Tract ◽  
Fluorometric Method ◽  
Maximal Inhibition ◽  
Fluorescent Intensity ◽  
E Coli ◽  
T24 Cells ◽  
Adhesion Activity ◽  
Tract Infections

Abstract Objectives Adhesion of type-P and type-1 fimbriated uropathogenic E. coli to urinary tract epithelial cells initiates urinary tract infections. This research aimed to optimize and apply a fluorometric method to evaluate the capacities of cranberry polyphenols and metabolites to inhibit such adhesion in vitro. Methods BacLight Green labelled E. coli were incubated with cranberry polyphenols or microbial metabolites of cranberry polyphenols for 30 min at 37°C. Mixture was added to a 96-well microplate containing 1 × 105/well of human uroepithelial T24 cells and incubated for 1 h at 37°C. After incubation, E. coli not adhered were removed by phosphate buffer washing. Fluorescent intensity was measured on a microplate reader at 480 nm excitation and 516 nm emission. Results Stable and strong fluorescent readings were obtained with 800 μmol/L BacLight Green for E. coli labeling and an E. coli to T24 cells ratio of 400:1 for co-incubation. A standard curve was established using 0–63 μM myricetin. The half-maximal inhibitory concentrations (IC50) of myricetin were 13.2 μM against type-P E. coli adhesion and 5.5 μM against type-1 E. coli adhesion. A fraction enriched with procyanidin polymers had IC50 of 57.6 μg/mL against type-P E. coli and 19.3 μg/mL against type-1 E. coli, respectively. Its anti-adhesion activities were more potent than those of cranberry fractions enriched with procyanidin oligomers, flavonols, or anthocyanin. Procyanidin A2 had a maximal inhibition about 35% at 17.3 μM against type-P E. coli, but no anti-adhesion activity was observed against type-1 E. coli. Procyanidin B2 showed a plateaued inhibition about 15% at 173–691 μM against type-P E. coli. Its maximal inhibition against type-1 E. coli was around 25% at 346 μM. Hippuric acid, a major metabolite of cranberry polyphenols, had a maximal inhibition about 20% at 558 μM against type-1 E. coli adhesion, whereas its anti-adhesion activity against type-P E. coli was not detected. Conclusions The optimized fluorometric method showed that both structure and composition of cranberry polyphenols and metabolites affected their abilities to inhibit E. coli adhesion in vitro. Anti-adhesion activities of cranberry polyphenols also depend on type of E. coli fimbriae. Funding Sources University of Florida Research Foundation Seed Fund.

scholarly journals In Vivo Phase Variation of Escherichia coli Type 1 Fimbrial Genes in Women with Urinary Tract Infection

1998 ◽  
Vol 66 (7) ◽  
pp. 3303-3310 ◽  
Author(s):  
Jean K. Lim ◽  
Nereus W. Gunther ◽  
Hui Zhao ◽  
David E. Johnson ◽  
Susan K. Keay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Acute Pyelonephritis ◽  
Urine Samples ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Tract Infections

ABSTRACT Type 1 fimbriae, expressed by most Escherichia colistrains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by SnaBI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First,E. coli strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with E. coli CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.

scholarly journals In Vitro Coliform Resistance to Bioactive Compounds in Urinary Infection, Assessed in a Lab Catheterization Model

2021 ◽  
Vol 11 (9) ◽  
pp. 4315
Author(s):  
Emanuel Vamanu ◽  
Laura Dorina Dinu ◽  
Cristina Mihaela Luntraru ◽  
Alexandru Suciu
Keyword(s):  
Urinary Tract ◽  
Bioactive Compounds ◽  
Urinary Tract Infections ◽  
Antimicrobial Effect ◽  
Biologically Active ◽  
Bacterial Strains ◽  
E Coli ◽  
Functional Product ◽  
Tract Infections

Bioactive compounds and phenolic compounds are viable alternatives to antibiotics in recurrent urinary tract infections. This study aimed to use a natural functional product, based on the bioactive compounds’ composition, to inhibit the uropathogenic strains of Escherichia coli. E. coli ATCC 25922 was used to characterize the IVCM (new in vitro catheterization model). As support for reducing bacterial proliferation, the cytotoxicity against a strain of Candida albicans was also determined (over 75% at 1 mg/mL). The results were correlated with the analysis of the distribution of biologically active compounds (trans-ferulic acid-268.44 ± 0.001 mg/100 g extract and an equal quantity of Trans-p-coumaric acid and rosmarinic acid). A pronounced inhibitory effect against the uropathogenic strain E. coli 317 (4 log copy no./mL after 72 h) was determined. The results showed a targeted response to the product for tested bacterial strains. The importance of research resulted from the easy and fast characterization of the functional product with antimicrobial effect against uropathogenic strains of E. coli. This study demonstrated that the proposed in vitro model was a valuable tool for assessing urinary tract infections with E. coli.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

scholarly journals Determination of nitrofurantoin and fosfomycin susceptibility among urinary Escherichia coli isolates

2020 ◽  
Vol 8 (9) ◽  
pp. 3274
Author(s):  
Rachana Kanaujia ◽  
Amit Kumar ◽  
Malay Bajpai
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Urine Samples ◽  
E Coli ◽  
Tract Infections ◽  
Therapeutic Alternatives ◽  
Micro Organisms ◽  
Staphylococcus Spp

Background: Urinary tract infections (UTIs) are one of the most common infections. For treatment of UTIs, there are limited antibiotics due to increased resistance among uropathogens. Two older antibiotics; Nitrofurantoin and Fosfomycin have become novel oral therapeutic options against uropathogens. Aim of the study was to identify UTI causing micro-organisms and evaluate in-vitro activity of nitrofurantoin and fosfomycin against most common isolated organism (E. coli).Methods: Results of urine samples culture and susceptibility testing over a period of 1 year were analysed and included in this study.Results: Micro-organisms were isolated from 568 urine samples. Most commonly isolated organism was Escherichia coli (40.50%), followed by Klebsiella spp. (20.07%) and Staphylococcus spp. (17.07%). Susceptibility of E. coli to nitrofurantoin and fosfomycin was 91.74% and 65.65% respectively. Conclusion: Good activity of nitrofurantoin and fosfomycin against E. coli indicates that these two drugs are potential therapeutic alternatives for urinary tract infections.

scholarly journals The Globoseries Glycosphingolipid Sialosyl Galactosyl Globoside Is Found in Urinary Tract Tissues and Is a Preferred Binding Receptor In Vitro for Uropathogenic Escherichia coli Expressing pap-Encoded Adhesins

1998 ◽  
Vol 66 (8) ◽  
pp. 3856-3861 ◽  
Author(s):  
A. E. Stapleton ◽  
M. R. Stroud ◽  
S. I. Hakomori ◽  
W. E. Stamm
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Blood Group Antigens ◽  
E Coli ◽  
Vaginal Epithelial Cells ◽  
History Of ◽  
Tract Infections

ABSTRACT Women with a history of recurrent Escherichia coliurinary tract infections (UTIs) are significantly more likely to be nonsecretors of blood group antigens than are women without such a history, and vaginal epithelial cells (VEC) from women who are nonsecretors show enhanced adherence of uropathogenic E. coli isolates compared with cells from secretors. We previously extracted glycosphingolipids (GSLs) from native VEC and determined that nonsecretors (but not secretors) selectively express two extended globoseries GSLs, sialosyl galactosyl globoside (SGG) and disialosyl galactosyl globoside (DSGG), which specifically bound uropathogenicE. coli R45 expressing a P adhesin. In this study, we demonstrated, by purifying the compounds from this source, that SGG and DSGG are expressed in human kidney tissue. We also demonstrated that SGG and DSGG isolated from human kidneys bind uropathogenic E. coli isolates expressing each of the three classes ofpap-encoded adhesins, including cloned isolates expressing PapG from J96, PrsG from J96, and PapG from IA2, and the wild-type isolates IA2 and R45. We metabolically 35S labeled these five E. coli isolates and measured their relative binding affinities to serial dilutions of SGG and DSGG as well as to globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4), two other globoseries GSLs present in urogenital tissues. Each of the five E. coli isolates bound to SGG with the highest apparent avidity compared with their binding to DSGG, Gb3, and Gb4, and each isolate had a unique pattern of GSL binding affinity. These studies further suggest that SGG likely plays an important role in the pathogenesis of UTI and that its presence may account for the increased binding of E. colito uroepithelial cells from nonsecretors and for the increased susceptibility of nonsecretors to recurrent UTI.

scholarly journals Uropathogenic Escherichia coli Triggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide

2004 ◽  
Vol 72 (8) ◽  
pp. 4570-4578 ◽  
Author(s):  
Robert Blomgran ◽  
Limin Zheng ◽  
Olle Stendahl
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
The Difference ◽  
Tract Infections ◽  
Neutrophil Survival

ABSTRACT Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of d-mannose. These results suggest that LPS is required for E. coli to exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.

scholarly journals The UbiI (VisC) Aerobic Ubiquinone Synthase Is Required for Expression of Type 1 Pili, Biofilm Formation, and Pathogenesis in Uropathogenic Escherichia coli

2016 ◽  
Vol 198 (19) ◽  
pp. 2662-2672 ◽  
Author(s):  
Kyle A. Floyd ◽  
Courtney A. Mitchell ◽  
Allison R. Eberly ◽  
Spencer J. Colling ◽  
Ellisa W. Zhang ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Biofilm Formation ◽  
Energy Deficit ◽  
Wild Type ◽  
Content Type ◽  
Anoxic Conditions ◽  
E Coli ◽  
Type 1 Pili ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC), which causes the majority of urinary tract infections (UTI), uses pilus-mediated adherence to initiate biofilm formation in the urinary tract. Oxygen gradients withinE. colibiofilms regulate expression and localization of adhesive type 1 pili. A transposon mutant screen for strains defective in biofilm formation identified theubiI(formerlyvisC) aerobic ubiquinone synthase gene as critical for UPEC biofilm formation. In this study, we characterized a nonpolarubiIdeletion mutant and compared its behavior to that of wild-type bacteria grown under aerobic and anoxic conditions. Consistent with its function as an aerobic ubiquinone-8 synthase, deletion ofubiIin UPEC resulted in reduced membrane potential, diminished motility, and reduced expression of chaperone-usher pathway pili. Loss of aerobic respiration was previously shown to negatively impact expression of type 1 pili. To determine whether this reduction in type 1 pili was due to an energy deficit, wild-type UPEC and theubiImutant were compared for energy-dependent phenotypes under anoxic conditions, in which quinone synthesis is undertaken by anaerobic quinone synthases. Under anoxic conditions, the two strains exhibited wild-type levels of motility but produced diminished numbers of type 1 pili, suggesting that the reduction of type 1 pilus expression in the absence of oxygen is not due to a cellular energy deficit. Acute- and chronic-infection studies in a mouse model of UTI revealed a significant virulence deficit in theubiImutant, indicating that UPEC encounters enough oxygen in the bladder to induce aerobic ubiquinone synthesis during infection.IMPORTANCEThe majority of urinary tract infections are caused by uropathogenicE. coli, a bacterium that can respire in the presence and absence of oxygen. The bladder environment is hypoxic, with oxygen concentrations ranging from 4% to 7%, compared to 21% atmospheric oxygen. This work provides evidence that aerobic ubiquinone synthesis must be engaged during bladder infection, indicating that UPEC bacteria sense and use oxygen as a terminal electron acceptor in the bladder and that this ability drives infection potential despite the fact that UPEC is a facultative anaerobe.

scholarly journals A Randomized, Double-Blind, Placebo-Controlled Pilot Study to Assess the Urinary Anti-Adhesion Activity Following Consumption of Cranberry + health™ Cranberry Supplement (P06-114-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Haiyan Liu ◽  
Christina Khoo
Keyword(s):  
Urinary Tract ◽  
Ex Vivo ◽  
Pilot Trial ◽  
Type I ◽  
Double Blind ◽  
Double Blind Placebo ◽  
E Coli ◽  
Adhesion Activity ◽  
Tract Infections ◽  
Time Point

Abstract Objectives Urinary tract infections (UTIs) are the second most common bacterial infections and are often treated with antibiotics. The North American cranberry is recommended for UTI prophylaxis in women with recurrent UTI as a nutritional alternative approach. The ability of cranberry components or their metabolites to inhibit the adhesion of uropathogenic E.coli to the bladder is considered as an important mechanism by which cranberry mitigates UTIs. The objective of this study was to evaluate the anti-adhesion activity of urine from healthy individuals against both type I pili and P-fimbriae uropathogenic E.coli, after the consumption of Cranberry + health™ Cranberry Supplement. Methods In this randomized, double-blind, placebo-controlled pilot trial (n = 20), subjects consumed two cranberry chews, one in the morning and one in the evening. Clean-catch urine samples collected at baseline and post-intervention (0–3 h, 3–6 h, 6–9 h, 9–12 h, 12–24 h, 24–30 h, 30–36 h) were tested for anti-adhesion activity utilizing both a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriae E. coli and a T24 cell line model for type I pili E.coli (UTI89). Results Urinary ex vivo anti-adhesion activity against P-fimbriae E. coli after consuming cranberry chew was significantly greater (p < 0.05) than placebo chew at 0–3 h, 3–6 h, 6–9 h, 9–12, and 12–24 time point. Urinary anti-adhesion activity against type I pili E.coli (UTI89) following cranberry chew was significantly greater (p < 0.1) than placebo chew at 3–6 h, 6–9 h, and 12–24 h time point. Conclusions Acute consumption of Cranberry + health™ Cranberry Supplement provides ex vivo anti-adhesion activity, which may help to improve urinary tract health. Funding Sources Ocean Spray Cranberries, Inc.

scholarly journals Escherichia coli CFT073 Fitness Factors during Urinary Tract Infection: Identification Using an Ordered Transposon Library

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Allyson E. Shea ◽  
Juan Marzoa ◽  
Stephanie D. Himpsl ◽  
Sara N. Smith ◽  
Lili Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Human Urine ◽  
Transposon Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACT Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB. Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field. IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.

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