Inhibition of adherence of multi-drug resistant E. coli by proanthocyanidin

Proanthocyanidin is commonly used for inhibiting urinary tract infection (UTI) of sensitive strains of Escherichia coli. The aim of this study was to investigate the effect of proanthocyanidin on adherence of uropathogenic multi-drug resistant E. coli to uroepithelial cells, which has not yet been investigated so far. Extracts of the purified proanthocyanidin were prepared from dried cranberry juice. Purity and structural assignment of proanthocyanidin was assessed using high performance liquid chromatography and ¹³C nuclear magnetic resonance spectroscopy, respectively. Subsequently, its affect on multi-drug resistant bacteria as well as quantification of anti-adherence bioactivity on human vaginal and bladder epithelial cells was appraised. Inhibition of adherence to an extent of about 70% with multi-drug resistant E. coli strains was observed on uroepithelial cell. The anti-adherence bioactivity of the proanthocyanidin was detected at concentrations of 10–50 μg/ml with significant bacteriuria. Probable proanthocyanidin through A-type linkages either combines to P-fimbriae of bacterial cells or modifies the structural entity of P-fimbriae and inhibits bacterial adherence to uroepithelial cells. The proanthocyanidin exhibited anti-adherence property with multi-drug resistant strains of uropathogenic P-fimbriated E. coli with in vitro study. Hence proanthocyanidin may be considered as an inhibitory agent for multi-drug resistant strains of E. coli adherence to uroepithelial cells.     

Adherence of gram-negative uropathogens to human uroepithelial cells

The adherence of several gram-negative uropathogens to human uroepithelial cells was examined with scanning and transmission electron microscopy, and in vitro adhesion assays. The bacteria studied demonstrated different extracellular structures: fimbria and glycocalyx. Human uroepithelial cells were obtained from a bladder tumor cell line (T-24) and from different groups of patients, including those with no history of urinary tract infection (controls), recurrent urinary tract infection, acute cystitis and bladder tumors before and after bacillus Calmette-Guerin therapy. Results from these comparative studies showed that bacteria with extracellular structures adhered better than those bacteria without extracellular structures. It also was shown that uroepithelial cells obtained from patients with recurrent urinary tract infection and with acute cystitis were more susceptible to bacterial adherence than the uroepithelial cells of the controls (there were significant increases of 2 to 5-fold in mean adherence in the former 2 groups, p equals 0.015 and 0.002). There was no significant difference in bacterial adherence between the T-24 cells and the bladder tumor cells before bacillus Calmette-Guerin treatment. However, in both groups after treatment with bacillus Calmette-Guerin the mean adherence increased 2 to 4-fold (p equals 3 times 10(-5)). A survey of 29 bladder tumor patients also showed a 5-fold increase in the incidence of acquired urinary tract infection after bacillus Calmette-Guerin therapy. These results reveal a correlation between bacterial adherence and urinary tract infection, and suggest that bacterial adherence to uroepithelial cells depends upon the bacterial extracellular structures and the source of the uroepithelial cells.     

Secretory immunoglobulin A and inhibitory activity of bacterial adherence to epithelial cells in urine from patients with urinary tract infections

Summary To assess the role of local immune response against bacterial invasion of the urinary tract we studied 168 patients with bacteriuria. Urinary secretory immunoglobulins A (sIgA) were measured using radial immunodiffusion or enzyme-linked immunosorbent assay (ELISA). In particular, ELISA is a very suitable assay for measuring the low levels of sIgA in urine. Furthermore, we used a quantitative in vitro adherence assay to investigate the attachment of Escherichia coli to human uroepithelial cells after incubation in urine from patients with urinary tract infection. Urine from patients with ileocystoplasty was significantly more potent in inhibiting bacterial adherence than was urine from other groups of patients with urinary tract infection. The presence of high urinary sIgA may help explain the increased antiadherence activity of urine in patients with ileocystoplasty. Mean urinary sIgA in patients with upper urinary tract infection was higher than in patients with uncomplicated infection in the lower urinary tract. Alterations in mucosal immune functions may account for the propensity toward bacterial colonization in women prone to uncomplicated urinary tract infection.     

Ability of uropathogens to bind to tamm horsfall protein-coated renal tubular cells

Summary Epithelial cells were isolated from a healthy human kidney and found to be of normal, renal tubular origin. The cells were maintained in tissue culture and found to secrete Tamm Horsfall protein (THP). Three strains of uropathogenic Escherichia coli, and one each of Proteus mirabilis and Pseudomonas aeruginosa were found to adhere to the THP-coated tubular cells. Extraneous THP bound to all the organisms except P fimbriated E. coli, and caused a reduction in the adhesion of type 1 fimbriated E. coli and P. mirabilis to the renal cells. However, irrespective of reduced adhesion in three of five strains tested, there was adequate evidence to indicate that THP does not competitively exclude all uropathogenic adhesion. On the contrary, its presence on renal cells may act to mediate bacterial colonization.     

Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence of gram-negative uropathogens by competitive exclusion

The adherence of the normal flora to human uroepithelial cells was examined using scanning and transmission electron microscopy and an in vitro adhesion assay. The normal flora were isolated from human cervical, vaginal and distal urethral surfaces. Human uroepithelial cells were obtained from healthy females (controls) and from female patients with recurrent urinary tract infection. The results indicate that: 1) there was no significant difference between the mean adherence of the normal flora to the uroepithelial cells from controls or patients, but there were quantitative differences between different bacterial species of the normal flora attaching to uroepithelial cells; 2) bacterial species isolated from the distal urethral and vaginal surfaces attached in larger numbers to uroepithelial cells from controls than from patients at day 10 of the menstrual cycle (peak value of adherence); 3) the adherence patterns of the cervical and vaginal microbial flora were bicyclic and appeared to be related to the human menstrual cycle; 4) complete or partial inhibition of adherence of several Gram-negative uropathogens was achieved by preincubating the uroepithelial cells with several bacterial species of the normal flora. Results from this study suggest that the normal flora of the urinary tract may play an important protective role against attachment of uropathogens to the surfaces of uroepithelial cells, and that the blocking capacity of these organisms may vary over the menstrual cycle.     

Adherence of uropathogenic E. coli to differentiated human uroepithelial cells grown in vitro

A quantitative in vitro model to measure E. coli adherence to differentiated human uroepithelial cells has been developed. Primary cultures of uroepithelial cells were initiated from normal ureteral epithelium. Adherence of uropathogenic 3H-labelled Escherichia coli to postconfluent human uroepithelial cells was directly related to the bacteria:epithelial cell ratio during incubation. Bacterial attachment was inhibited either by mannose or by urine containing anti-E. coli antibodies. Transmission electron microscopy showed that epithelial cells differentiated in vitro to resemble normal uroepithelium in vivo. Furthermore, electron microscopy showed specific adherence of bacteria to the glycocalyx of microvilli of the superficial uroepithelial cells in vitro in a manner which closely mimics the in vivo interaction. This model of bacterial adherence permits in vitro analysis of adhesin-receptor interactions between uropathogenic E. coli and a layer of viable uroepithelial cells similar to those lining the bladder.     

Urinary tract infections and their prevention

ContextThis article reviews diverse aspects of the prevention of urinary tract infections, including confirmation of the diagnosis, application of hygiene and dietary measures, antibacterial prophylaxis (preferably consisting of a single nocturnal oral dose per day of an antibiotic or drug with high urinary excretion and good tolerance), and administration of vaccines made with Escherichia coli and other Gram-negative bacilli, consisting of immunostimulating fractions of E. coli strains or E. coli type-1 fimbriae administered through the parenteral or oral route.ObjectiveWe aimed to review the new preventive measures against urinary tract infections.Acquisition and synthesis of evidenceWe reviewed various microbiological aspects, as well as the physiopathology and virulence factors of uropathogenic E. coli strains expressing type-1 and P fimbriae. The association between blood groups and urinary tract infections in blood group antigen-secretors and nonsecretors was analyzed.ConclusionsNew preventive measures against urinary tract infection consist of the use of phenol-inactivated vaccines administered via the mucosal route, inhibitors of bacterial adherence and biofilm formation and cyclic adenosine monophosphate stimulators, especially in women aged between puberty and menopause, who show the highest incidence of these infections.     

Factors predisposing to urinary tract infections in children

The normal urinary tract is sterile for many reasons. These include: bacterial eradication by urinary and mucus flow, urothelial bactericidal activity, urinary secretory IgA, and blood group antigens in secretions which interfere with bacterial adherence. Periodic emptying of the bladder should wash bacteria out, but uropathogens grow well in urine and their rapid doubling time might not clear bacteria by voiding at the usual frequency. The ability to colonize the gut, as well as adhere to host squamous and urothelial cells, is due to bacterial adherence, a mechanism by which fluid washout would not be effective. Fimbriae or pili, hair-like surface appendages of Escherichia coli, are the usual adhesins. E. coli with type 1 fimbriae adhere to mucus, and P-fimbriae adhere to glycolipids on mucous membranes and urothelial cells. Other common virulence factors of E. coli include the capsule, which prevents phagocytosis, hemolysin, which damages urothelium, and aerobactin, which sequesters iron. Adherence stimulates the inflammatory response by activation of cytokines such as interleukin-1, -6, and -8. These cytokines stimulate the production of intercellular adhesion molecule, which by leukocyte adhesion causes migration of these cells to the site of infection to counteract it. Received September 22, 1995; received in revised form January 22, 1996; accepted January 25, 1996     

Host defense within the urinary tract. I. Bacterial adhesion initiates an uroepithelial defense mechanism

Uroepithelial defense has been suggested to contribute to the local host resistance against ascending urinary tract infection. The cellular mechanism, however, is not known. In this study, bacterial growth was measured under the direct and indirect influence of uroepithelial cells. To study if a specific ligand-receptor interaction is required for uroepithelial cell (UEC) activation, isogenic Escherichia coli mutants expressing either mannose-sensitive, mannose-resistant (p), or mannose resistant (s) pili were tested for their capacity to induce the UEC defense mechanism. The antibacterial effect of UEC was abolished either by performing coculture in chambers with a fluid-permeable membrane which separates UEC from bacteria or by inhibiting membrane contact using the antiadherence factor pentosane polysulfate. No difference between the various types of pili could be shown. All E. coli strains adhered comparably to the UEC and were subsequently suppressed in their growth. Even a “naked” mutant without expression of common pili showed a similar behavior. In conclusion, bacterial growth suppression depends on direct contact between the UEC and bacteria, but is independent of common pili expressed on E. coli. Received July 29, 1995; received in revised form and accepted December 28, 1995     

The influence of growth media on the morphology and in vitro adherence characteristics of gram-negative urinary pathogens

We studied the adherence of 3 groups of gram-negative urinary pathogens, grown in different culture media, to human uroepithelial cells obtained from healthy females (controls) and from female patients with recurrent urinary tract infection, using an in vitro adhesion assay and electron microscopy. Morphologic examination and mannose sensitivity testing of the uropathogens grown in urine showed that they expressed extracellular structures similar to those found in infected urine in vivo. However, the production of various bacterial extracellular structures responsible for adhesion can be enhanced or decreased when the same uropathogens are grown in media other than urine, resulting in an alteration of the mean adherence of the uropathogens to uroepithelial cells. Brain Heart Infusion broth enhances the production of fimbriae, Modified Vogel and Bonner's Medium enhances the production of exopolysaccharides, and yeast nitrogen base decreases the production of both. These studies suggest that bacterial extracellular structures should be examined morphologically prior to in vitro adhesion assays and that urine is the medium of choice for studying the adherence characteristics of various urinary pathogens in vitro.     

Cranberry products inhibit adherence of p-fimbriated Escherichia coli to primary cultured bladder and vaginal epithelial cells

PURPOSE: Cranberry proanthocyanidins have been identified as possible inhibitors of Escherichia coli adherence to uroepithelial cells. However, little is known about the dose range of this effect. Furthermore, it has not been studied directly in the urogenital system. To address these issues we tested the effect of a cranberry powder and proanthocyanidin extract on adherence of a P-fimbriated uropathogenic E. coli isolate to 2 new urogenital model systems, namely primary cultured bladder epithelial cells and vaginal epithelial cells. MATERIALS AND METHODS: E. coli IA2 was pre-incubated with a commercially available cranberry powder (9 mg proanthocyanidin per gm) or with increasing concentrations of proanthocyanidin extract. Adherence of E. coli IA2 to primary cultured bladder epithelial cells or vaginal epithelial cells was measured before and after exposure to these products. RESULTS: Cranberry powder decreased mean adherence of E. coli IA2 to vaginal epithelial cells from 18.6 to 1.8 bacteria per cell (p <0.001). Mean adherence of E. coli to primary cultured bladder epithelial cells was decreased by exposure to 50 mug/ml proanthocyanidin extract from 6.9 to 1.6 bacteria per cell (p <0.001). Inhibition of adherence of E. coli by proanthocyanidin extract occurred in linear, dose dependent fashion over a proanthocyanidin concentration range of 75 to 5 mug/ml. CONCLUSIONS: Cranberry products can inhibit E. coli adherence to biologically relevant model systems of primary cultured bladder and vaginal epithelial cells. This effect occurs in a dose dependent relationship. These findings provide further mechanistic evidence and biological plausibility for the role of cranberry products for preventing urinary tract infection.     

Viable but nonculturable uropathogenic bacteria are present in the mouse urinary tract following urinary tract infection and antibiotic therapy

Involvement of the viable but nonculturable (VBNC) condition in recurrent urinary tract infections (UTIs) was investigated. VBNC bacteria are those which are alive but do not give rise to visible growth under nonselective growth conditions. Urine, bladder, and kidney samples collected over a 2-month period from BALB/c mice inoculated with the uropathogenic Escherichia coli strain J96 were examined to determine the level of culturable and viable bacteria. Urine from uninoculated mice was found to contain more viable than culturable bacteria. Inoculated mice had a transient increase in the level of culturable forms of the uropathogen in their urine, followed by a decrease to background levels; they also had multiple log higher levels of viable cells than culturable cells. The culturable pathogenic bacteria in mice that were inoculated and received antibiotic treatment dropped to undetectable levels within 1 week. At 2 out of 12 subsequent time points spanning an additional 65 days, culturable forms of the inoculated pathogenic bacteria were recovered. Polymerase chain reaction (PCR) analysis confirmed that DNA from the inoculated bacteria was present in a sample that yielded no culturable bacteria. These data indicate that the inoculated uropathogenic E. coli was not eliminated by antibiotic therapy, and suggest that these bacteria may escape detection by current standard culturability assays because they are VBNC. Received: 24 March 2000 / Accepted: 11 September 2000     

Nitric Oxide Inhibits Neutrophil Adhesion during Experimental Extracorporeal Circulation

Background: Myocardial and pulmonary injuries often occur after cardiopulmonary bypass, mediated in part by neutrophil activation and adhesion to endothelial cells. The effects of nitric oxide (NO) administration on neutrophil adhesion to endothelial cells after simulated extracorporeal circulation were investigated.

Methods: Two identical extracorporeal circulation circuits were primed with fresh human blood and circulated for 2 h at 37 [degree sign]C. Nitric oxide at a 40-ppm concentration was added to one of the oxygenators in each pair. Neutrophil CD11b/CD18 expression and their adhesion to human umbilical vein endothelial cell monolayers were assayed in leukocytes isolated from samples drawn from the circuit 30, 60, 90, and 120 min after circulation began. In another series of experiments, blocking monoclonal antibodies to both neutrophil CD11b and CD18 were incubated with polymorphonuclear leukocytes after removal from the circuit before the adhesion assay.

Results: After 60 min of circulation, the neutrophils from NO-treated circuits showed significantly reduced CD11b/CD18 surface expression compared with the control group. There was also a significant reduction in neutrophil - endothelial adhesion in the NO group after 120 min of circulation. Monoclonal antibodies to both CD11b and CD18 significantly inhibited the adhesion of polymorphonuclear leukocytes at endothelial cells after 120 min of circulation.     

Influence of urological irrigation fluids on urothelial bacterial adherence

Summary The influence of various urological irrigation solutions on bacterial growth and adherence to urothelium was investigated in in vitro and guinea pig models. The irrigation solutions glycine 1.5%, glycine 1.5% and ethanol 1%, glycerol 3%, mannose 6%, sorbitol 2.7% and mannitol 0.54% all inhibited bacterial growth compared with normal saline. In guinea pigs, the influence on bacterial adherence of four irrigation solutions (glycine 1.5%, glycine 1.5% and ethanol 1%, mannose 6%, povidone-iodine) was investigated using two different strains of E. coli. After cauterizing one side of the bladder and inoculation with 2.7×10⁸ colony forming units under high or low pressure, the bladder was irrigated with the irrigation solutions. There was a stronger adherence of E. coli O6 (with type I pili) than of E. coli ATCC 25922 (without type I pili) to bladder urothelium, particularly to the injured side. There was no significant difference between the high- and low-pressure groups. None of the various irrigation solutions was clearly superior. As mannose 6% effectively inhibited type I pili and also had some antibacterial activity it may reduce urinary tract infection if used as irrigation solution.     

Increased risk of urinary tract infection associated with the use of calcium supplements

Summary Since ions are known to influence the interaction between cells, we undertook an examination of the effect of various ions on bacterial adherence to uroepithelial cells. While most of the ions examined had no effect or decreased bacterial adherence, calcium ions significantly increased bacterial adherence. It was demonstrated, in vitro that as the concentration of calcium was increased to levels higher than normally found in the urine, there was a significant increase in bacterial adherence. It was also found that if the diet was supplemented with calcium there was an increase in the excretion of calcium in the urine and a corresponding increase in bacterial adherence when bacteria and uroepithelial cells were incubated in this urine. It is suggested that an excretion of excess calcium in the urine may lead to increased bacterial adherence in vivo and an increased potential for urinary tract infections.     

Reduction of Escherichia coli adherence to uroepithelial bladder cells after consumption of cranberry juice: a double-blind randomized placebo-controlled cross-over trial

To determine the efficacy of the consumption of cranberry juice versus placebo with regard to the presence of in vitro bacterial anti-adherence activity in the urine of healthy volunteers. Twenty healthy volunteers, 10 men and 10 women, were included. The study was a double-blind, randomized, placebo-controlled, and cross-over study. In addition to normal diet, each volunteer received at dinner a single dose of 750 ml of a total drink composed of: (1) 250 ml of the placebo and 500 ml of mineral water, or (2) 750 ml of the placebo, or (3) 250 ml of the cranberry juice and 500 ml of mineral water, or (4) 750 ml of the cranberry juice. Each volunteer took the four regimens successively in a randomly order, with a washout period of at least 6 days between every change in regimen. The first urine of the morning following cranberry or placebo consumption was collected and used to support bacterial growth. Six uropathogenic Escherichia coli strains (all expressing type 1 pili; three positive for the gene marker for P-fimbriae papC and three negative for papC), previously isolated from patients with symptomatic urinary tract infections, were grown in urine samples and tested for their ability to adhere to the T24 bladder cell line in vitro. There were no significant differences in the pH or specific gravity between the urine samples collected after cranberry or placebo consumption. We observed a dose dependent significant decrease in bacterial adherence associated with cranberry consumption. Adherence inhibition was observed independently from the presence of genes encoding type P pili and antibiotic resistance phenotypes. Cranberry juice consumption provides significant anti-adherence activity against different E. coli uropathogenic strains in the urine compared with placebo.     

Preliminary study on the prevention of recurrent urinary tract infection in adult women using intravaginal Lactobacilli

We now report the development of gelatin suppositories containing freeze dried Lactobacillus casei GR-1 and L. fermentum B-54 chosen for their ability to adhere to uroepithelial cells and inhibit the growth of E. coli and Enterococcus faecalis in vitro. The product has a long shelf life, and is easily and safely utilized. In a preliminary study of 10 female patients who received the therapy at weekly intervals for 12 months, there was a net reduction of 66.3% in the incidence of recurrent urinary tract infections. Lactobacilli were isolated from the vagina of the patients for the duration of treatment. Examination of random specimens showed some degree of correlation between numbers of lactobacilli adherent to vaginal epithelial cells and viable organisms recovered from swabs.     

Adherence of gram-negative uropathogens to human uroepithelial cells

The ability of bacteria to adhere to the mucosal surface is generally regarded as an important factor in infection. In particular, most urinary tract pathogens reach the kidney from the bladder. The attachment to the mucosa may promote colonization of the ureters and penetration of the renal pelvis by ascending infection. We have examined 50 patients with urinary tract infection and the uropathogens isolated from their urines were tested for adhesion to epithelial cells from fresh morning urine of healthy women. The results confirm that adherence of the microorganisms to uroepithelial cells is important for the establishment of infections of the upper urinary tract.