A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Bacterial aggregation in sepsis

Previous studies have provided evidence that bacterial aggregation occurs in disease processes. In the present report, an assay system was used to demonstrate bacterial aggregation and coaggregation in vitro. The results showed that uropathogens aggregated in urine and saline and formed aggregates attached to uroepithelial cells. The presence of fimbriae and O, K, and H antigens on the cell surface did not appear to correlate with aggregation. Additional studies related to intraabdominal sepsis showed aggregation of aBacteroides fragilis strain and coaggregation betweenBacteroides fragilis andEscherichia coli, suggestive of in vivo interaction. Electron microscopy demonstrated the various aggregation reactions and was particularly effective in showing type 1 fimbriatedEscherichia coli coaggregating with theBacteroides fragilis. The ability of organisms to aggregate and coaggregate appears to have potential significance in health and disease.     

Effect of antibiotic treatment on receptivity of uroepithelial cells to uropathogens

The management of female patients with recurrent urinary tract infections still remains a problem, and long-term prophylactic or short intermittent courses of antibiotics are the standard forms of therapy. In this report, 10 patients were examined for the effects of long- and short-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX) antibiotics on the receptivity of uroepithelial cells to bacterial adherence. The urine of all patients was sterile while on antibiotic therapy. Few bacteria were found adherent to the cells from adult patients (group 1, mean age 36 years) on long-term antibiotics, but the cells were highly receptive to uropathogens in vitro, especially for Escherichia coli expressing mannose-resistant adhesins. Controls of age-matched adult females were included and in vitro adherence levels were found to be higher for those women with a history of urinary tract infection compared with those with no past record of infection. In the second group, elderly patients (mean age 87 years) presented with bacteriuria, and their uroepithelial cells were found to be colonized by uropathogens to a significantly greater extent than their controls. The adherent population was reduced during 7-day TMP-SMX antibiotic treatment, but increased posttherapy, particularly in two patients who subsequently became reinfected. The in vitro results showed that uroepithelial cells retain their receptivity to uropathogenic adherence, both during and after treatment. Although antibiotics eradicate uropathogens from the urinary tract, patients remain susceptible to recolonization by uropathogens and are at risk of reinfection after completion of therapy.     

Inhibition of bacterial adherence to rat bladder epithelial cells by human immune serum globulin

The ability of commerical human immune serum globulin (HISG) to inhibit the adherence of urinary tract infection isolates to rat bladder epithelial cells was investigated utilizing an in vitro adherence system. Significant decreases in adherence were noted when strains ofEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, andEnterobacter cloacae were tested against five HISG preparations. An enzyme-linked immunosorbent assay indicated that all five HISG preparations also contained antibodies against type-1 pili isolated fromKlebsiella pneumoniae. The presence of antibodies directed against a bacterial adhesin and the effectiveness of HISG in inhibiting the attachment of a wide range of urinary pathogens to bladder cells suggest that HISG may have practical therapeutic values in the prophylaxis of diseases where bacterial adherence is a prerequisite for the initiation of infection.     

In Vitro Evaluation of the Efficacy of a Silver-Coated Catheter

Bacteria commonly associated with nosocomial urinary tract infections were examined in vitro for their relative adherence to latex, 100% silicone-, hydrogel-coated latex-, and hydrogel/silver-coated latex urinary catheters. Degrees of adherence within 2 h were determined with cells radiolabeled with leucine. Adherence was greatest and equivalent on silicone and latex catheters. Adherence of four strains of Escherichia coli to the hydrogel/silver-coated catheter was decreased by 50% to 99% in comparison with the other catheters. Repeat testing with strains of E. coli and Pseudomonas aeruginosa with over 50 catheters demonstrated a consistency in the inhibition. The hydrophilic coating of the catheter appeared to be primary in the decreased adherence phenomenon followed by a secondary biocidal effect of the silver ion. Received: 2 December 1995 / Accepted: 3 January 1996     

Glycolipid depletion in antimicrobial therapy

Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N-butyldeoxynojirimycin (NB-DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P-fimbriated Escherichia coli. NB-DNJ blocks the ceramide-specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose-dependent way, and depletion markedly inhibited P-fimbriated bacterial attachment in vitro. NB-DNJ-fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P-fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae-mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.     

Characteristics of Biofilms from Urinary Tract Catheters and Presence of Biofilm-Related Components in Escherichia coli

Long term catheterization of the urinary tract leads to bacterial colonization of the urine, whereby adherence to the catheter surface is a major determinative factor for colonization. Collection of bacterial isolates from urine and urinary catheters of 45 patients showed multi-species catheter-colonization, while Escherichia coli isolates were frequently found in the urine in high numbers. Biofilm formation of catheter and urine-derived E. coli isolates was associated with the presence of the fluA gene, loss of O-antigen, and expression of type 1 fimbriae. The second messenger cyclic di-GMP (cdiGMP), a major regulator of biofilm formation, regulated adherence to the catheter surface in a selected clinical isolate suggesting that the cdiGMP second messenger pathway may be a target for anti-biofilm therapeutic approaches.     

Adherence to uroepithelial cells of Providencia stuartii isolated from the catheterized urinary tract

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.     

Preventive and therapeutic administration of an indigenous <Emphasis Type="Italic">Lactobacillus</Emphasis> sp. strain against <Emphasis Type="Italic">Proteus mirabilis</Emphasis> ascending urinary tract infection in a mouse model

Probiotics are increasingly being considered as non-pharmaceutical and safe potential alternatives for the treatment and prevention of a variety of pathologies including urinary tract infections. These are the most common infections in medical practice and are frequently treated with antibiotics, which have generated an intense selective pressure over bacterial populations. Proteus mirabilis is a common cause of urinary tract infections in catheterised patients and people with abnormalities of the urinary tract. In this work we isolated, identified and characterised an indigenous Lactobacillus murinus strain (LbO2) from the vaginal tract of a female mouse. In vitro characterisation of LbO2 included acid and bile salts tolerance, growth in urine, adherence to uroepithelial cells and in vitro antimicrobial activity. The selected strain showed interesting properties, suitable for its use as a probiotic. The ability of LbO2 to prevent and even treat ascending P. mirabilis urinary tract infection was assessed using an experimental model in the mouse. Kidney and bladder P. mirabilis counts were significantly lower in mice preventively treated with the probiotic than in non-treated mice. When LbO2 was used for therapeutic treatment, bladder counts of treated mice were significantly lower although no significant differences were detected in P. mirabilis kidney colonisation of treated and non-treated animals. These results are encouraging and prompt further research related to probiotic strains and the basis of their effects for their use in human and animal health.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

Pathogenesis of urinary tract infection in the elderly: the role of bacterial adherence to uroepithelial cells

The in vitro adherence of nine strains of Gram-negative bacteria to uroepithelial cells from 24 women patients (>65 years) was significantly higher than to cells from 24 premenopausal women (18–40 years). Uroepithelial cells from patients with a history of previous urinary tract infection (UTI) were marginally more receptive to attachment of uropathogens than cells from women without a history of UTI, but this was not statistically significant. Serum from four elderly women with asymptomatic bacteriuria was used to stabilize samples for electron-microscopic examination, which showed the presence of fibrous glycocalyx material surrounding the bacteria and attached to the uroepithelial cells. Eighty uropathogenic isolates from elderly and premenopausal women were found to express adhesins, to produce urease and hemolysins, and to ferment sucrose, salicin, and dulcitol. These results suggest that the increased receptivity of uroepithelial cells to bacterial attachment may be a predisposing factor in the onset of UTI in the hospitalized and domiciliary elderly population.     

Functional analysis of the minor subunits of S fimbrial adhesion (SfaI) in pathogenic Escherichia coli

S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and -H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site-directed mutagenesis. Received: 15 March 1999 / Accepted: 2 November 1999     

Extraction and properties of nonfimbrial mannose-resistant hemagglutinin from a urinary isolate ofEscherichia coli

A mannose-resistant hemagglutinin (MRH) was extracted from an agar-grown urinary isolate ofEscherichia coli (827) by heating the organisms at 65°C for 1 h. Both MRH and the organisms agglutinated erythrocytes from human (group A) but not from seven other animal species tested, but were devoid of any detectable fimbriae as examined by electron microscopy. MRH was sensitive to pronase or 100°C heating, but not to trypsin or periodate, could not be sedimented by ultracentrifugation 149,000g, and passed the void volume of Sepharose-4B column. MRH inhibited the adherence of bacteria to tissue culture cells. The results suggest thatE. coli human isolate produces mannose-resistant hemagglutinin, which is located on the cell surface in nonfimbrial form and probably mediates adherence of the bacteria to eukaryotic cells.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

Production of the Escherichia coli Common Pilus by Uropathogenic E. coli Is Associated with Adherence to HeLa and HTB-4 Cells and Invasion of Mouse Bladder Urothelium

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract.     

A Novel Role for D-Alanylation of Lipoteichoic Acid of Enterococcus faecalis in Urinary Tract Infection

Background

Enterococci are the third most common cause of healthcare-associated infections, which include urinary tract infections, bacteremia and endocarditis. Cell-surface structures such as lipoteichoic acid (LTA) have been poorly examined in E. faecalis, especially with respect to urinary tract infections (UTIs). The dlt operon is responsible for the D-alanylation of LTA and includes the gene dltA, which encodes the D-alanyl carrier protein ligase (Dcl). The involvement of LTA in UTI infection by E. faecalis has not been studied so far. Here, we examined the role of teichoic acid alanylation in the adhesion of enterococci to uroepithelial cells.

Results

In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔdltA mutant colonizes uroepithelial surfaces more efficiently than wild type bacteria. We also demonstrated that this mutant adhered four fold better to human bladder carcinoma cell line T24 compared to the wild type strain. Bacterial adherence could be significantly inhibited by purified lipoteichoic acid (LTA) and inhibition was specific.

Conclusion

In contrast to bacteraemia model and adherence to colon surfaces, E. faecalis 12030ΔdltA mutant colonized uroepithelial surfaces more efficiently than wild-type bacteria. In the case of the uroepithelial surface the adherence to specific host cells could be prevented by purified LTA. Our results therefore suggest a novel function of alanylation of LTA in E. faecalis.     

Iron dictates the virulence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in urinary tract infections

Iron-limiting conditions have been reported to be prevalent in the milieu of urinary tract. In the present investigation, effect of iron on virulence of uropathogenic Pseudomonas aeruginosa in planktonic and biofilm cell mode was studied. Significant enhancement in elaboration of all the virulence traits along with increased adherence to uroepithelial cells and decreased phagocytosis of P. aeruginosa was observed following growth in iron-deplete medium. On the contrary, decrease in all these parameters except phagocytosis was observed when P. aeruginosa was grown in iron-rich medium. In vivo, P. aeruginosa grown in iron-deplete medium showed increased renal bacterial load and tissue pathology in a mouse model of ascending urinary tract infection compared with organisms grown in iron-replete medium. The results of the present study may help in understanding host–parasite interaction and in developing alternative preventive approach against P. aeruginosa induced urinary tract infections.     

Characterization of adhesion associated surface properties of uropathogenicEscherichia coli

Escherichia coli was isolated from the urine of patients with pyelonephritis, with urinary tract infections other than pyelonephritis and with asymptomatic bacteriuria. Surface properties of the strains were analyzed by the salting-out aggregation test (SAT), hydrophobic interaction chromatography (HIC), Congo red binding (Crb), agglutination of erythrocytes (MRHA) and latex particles covered by digalactoside (PF) and by adherence to tissue culture cells. In addition, a DNA probe for thepap gene was used. The DNA probe detected the highest proportion of strains withpap gene in the group of patients with pyelonephritis, lower in the urinary tract infections other than pyelonephritis and the lowest in the group with asymptomatic bacteriuria. Tests for P-fimbriae (PF, MRHA) showed a similar distribution. Hydrophobicity measured by SAT and by HIC did not show differences among the tested groups of strains. The results suggest that factors other than the P-fimbriae and hydrophobicity may contribute to the persistence ofE. coli in the urinary tract.     

Contribution of Tamm-Horsfall protein to virulence of Pseudomonas aeruginosa in urinary tract infection

Tamm-Horsfall glycoprotein (THP) is the most abundant protein which is synthesized by renal tubular cells and excreted in urine. Its role in urinary tract infection has yet not been identified. In the present study, the contribution of THP towards adherence of Pseudomonas aeruginosa to uroepithelial cells and murine peritoneal macrophages was studied. Decreased adherence of THP-coated P. aeruginosa to UECs and phagocytes was observed in vitro. In vivo, P. aeruginosa showed increased renal bacterial load and tissue pathology in a mouse model of acute ascending pyelonephritis, when THP-coated P. aeruginosa was used to cause infection. This study shows that THP may not necessarily act as a host defense component; rather, it may help in renal colonization of P. aeruginosa in vivo.