The human antibody response to uropathogenic Escherichia coli: a review

Urinary tract infections caused by Escherichia coli are associated with a local and systemic antibody response. We have studied the serum and urine antibody responses to Escherichia coli in men and women with pyelonephritis, cystitis, and asymptomatic bacteriuria. Protein immunoblots consistently demonstrated serum antibody response to lipopolysaccharide (LPS). Anti-LPS antibody titres rose significantly and progressively when comparing acute with convalescent sera in those who have had their first urinary infection. For those with repeated infections, high titre LPS antibodies were present and did not change significantly between acute and convalescent sera. Antibody responses to the major outer membrane proteins were present but did not differ significantly when compared with normal human serum. A specific anti-P pilus antibody response was demonstrated by immunoblotting. Anti-P pilus antibody was quantitated using ELISA and the titres were found to be very low. Three other techniques were also used to demonstrate the presence of serum antibody. Antibody was detectable by immunofluorescence, but the antigenic specificity of the antibody was more difficult to ascertain. Immunoprecipitation was more specific for determining the nature of the antibody response. Lastly, immunoelectron microscopy was valuable in demonstrating antipilus and antiflagellar antibodies. Immunoelectron microscopy and immunoblotting provided evidence that human antiserum to P pili was modestly cross-reactive and could bind heterologous P pili. These studies indicated that the major antibody response in humans occurs after pyelonephritis and is directed against LPS. An anti-P pilus response is frequently present and is cross-reactive to some extent with other P pili.     

Antipili antibody affords protection against ascending pyelonephritis in rat: evaluated by renal brush border membrane enzymes

Kinetic parameters (Km and Vmax) of renal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, leucine aminopeptidase and gamma-glutamyltranspeptidase were worked out in control, infected and immunized-infected rats. There was no significant change in the Km of all the enzymes studied in three groups. The Vmax of all the enzymes studied decreased significantly (p less than 0.05) 3 or 4 days postinfection and onwards in the left obstructed kidney of infected and immunised-infected animals. However, in the right unobstructed kidney the Vmax of alkaline phosphatase and leucine aminopeptidase increased significantly (p less than 0.05) in the early stages and decreased (p less than 0.05) in later stages in both the experimental groups. The significant difference (p less than 0.05) in the Vmax of infected and immunized-infected groups at various stages of infection revealed the partial protective role of antipili antibody against ascending pyelonephritis.     

Escherichia coli-specific T lymphocytes in experimental pyelonephritis

We have assessed the phenotype and specificity of infiltrating mononuclear cells in a model of unilateral ascending acute pyelonephritis induced in rats with nephritogenic Escherichia coli or Pseudomonas aeruginosa. Histologic examination showed a predominance of mononuclear cells in the interstitium at all periods examined (4, 8, 15, 21, and 25 days), although at 4 and 8 days neutrophils were also abundant. Most of the mononuclear cells had the morphologic appearance of large lymphocytes. Immunoperoxidase studies with mAb showed that most of the mononuclear cells were W3/25+; many were W3/13+ and a small proportion were OX8+. Many of the mononuclear cells were Ia+. T cells were propagated in IL-2-containing media from small fragments of renal tissue with pyelonephritic lesions. Most of the propagated cells were W3/25+; fewer than (10%) were OX8+ or Ia+. T cells propagated from kidneys infected with E. coli responded, in proliferation assays, to the infecting strain or other E. coli strains, but not to P. aeruginosa or enterococci. The response to non-p-pilus-bearing E. coli was as great or greater than to E. coli with adhesins. T cells derived from lesions induced by P. aeruginosa responded to the infecting organisms, but not to E. coli. The response to the infecting organism (E. coli or P. aeruginosa) was MHC restricted, as indicated by the requirement for syngeneic APC. The results show that large numbers of T lymphocytes, especially with the "helper/inducer" phenotype, accumulate in the lesions of acute pyelonephritis in rats. Among the infiltrating T lymphocytes are activated cells and cells with specific reactivity to the infecting bacteria (or related strains). The findings indicate that T lymphocytes play a role within the kidney in response to the invading bacteria.     

Pyelonephritis Therapy in Rats: Random Association with Excreted Urinary Antibacterial Activity

In vitro activities or excreted urinary activities of 56 generic and experimental drugs against two organisms commonly associated with human pyelonephritis, Escherichia coli and Proteus mirabilis, are not correlated with the in vivo activities of these compounds as measured by reduction of viable organisms in kidneys in experimental pyelonephritis.     

Serum antibodies to commensal oral and gut bacteria vary with age

Abstract Pyelonephritis is the most common urinary tract infection affecting females of all age groups. Despite concerted efforts the mechanism of renal injury in pyelonephritis is not clearly understood. In the present study we have made an attempt to characterise the mediators of inflammatory insult in an experimental model of ascending pyelonephritis. Mice infected with Escherichia coli O6:K13:H1 were sacrificed at 2, 7 and 14 days post-infection. Luminol-dependent chemiluminescence response, NADPH oxidase, acid phosphatase, β-glucuronidase and N -acetyl-β- d -glucosaminidase activities were monitored in circulating as well as renal phagocytic cells in order to determine the role of reactive oxygen species and lysosomal enzymes in genesis of renal injury. We have demonstrated that reactive oxygen species are generated at the initiation of infection and the levels increase progressively during the course of infection. While intracellular release of lysosomal enzymes was seen in all groups, extracellular release was primarily observed at 7 and 14 days post-infection only. The results indicate that while reactive oxygen species play a significant role in tissue injury during all stages of infection, lysosomal enzyme release in extracellular milieu augments tissue destruction at later stages only.     

Demonstration of bacterial antigen in macrophages in experimental pyelonephritis

The immunomorphological characteristics of interstitial macrophages with PAS-positive granules were studied in experimental Escherichia coli pyelonephritis in rats, using an anti-E. coli antibody. Immunohistochemical, immunoelectron microscopical, as well as light- and electron microscopical findings were compared at twelve time points between 2 days and 13 weeks after infection. Macrophages with PAS-positive granules were present in the inflammatory infiltrates from the 7th day. The granules were phagolysosomes, filled predominantly with myelin figures. The myelin figures originated mainly from the constituents of the bacterial wall and reacted with the anti-E. coli antibody even 13 weeks after infection. The storage of bacterial residues with preserved antigenic structures for several weeks after infection indicates disturbed phagolysosomal elimination of the bacterial substances in the PAS-positive macrophages. In the formation of macrophages with PAS-positive granules, lysosomal overloading with large amounts of bacteria and cell debris is assumed, leading to consumption of the lysosomal enzymes, consequent incomplete breakdown and retention of the bacterial residues.     

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD_cl4 cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.     

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.     

Etiology of acute and chronic pyelonephritis in children in Khabarovsk region

Microflora of urinary tract was studied in 419 children aged 1 - 17 years and hospitalized due to acute or chronic pyelonephritis. Etiology of inflammatory process was established in 57.8% of cases. According to our study, etiologic structure of causative agents of pyelonephritis did not differ from all-Russian data. The leading positions belonged to Gram-negative microorganisms from Enterobacteriaceae family: Escherichia coli, Proteus mirabilis, and Klebsiella spp. Results of the study point to high susceptibility of main causative agents of pyelonephritis to cephalosporins, aminoglycosides, and fluoroquinolones. High resistance to aminopenicillines was noted. In several isolates from Enterobacteriaceae family significant resistance to nalidixic acid and furazidin was observed.     

Significance of the ability of E. coli to alter capillary permeability for the reproduction of pyelonephritis in mice]

Experiments in a number of biological models showed that the cultures of E. coli isolated from the urine of children with pyelonephritis had a varied spectrum of pathogenic properties. Histologically confirmed pyelonephritis induced by intravenous infection in CBA mice, treated with 5% glucose by the method of Montgomerie et al., correlated with the bacterial adhesiveness to the epithelium and the interference with capillary permeability, registered in experiments on the pulmonary model with Evans blue used for control. The criterion for the development of pyelonephritis in mice, which was based, in the opinion of Mintogemerie et al., on the positive results, indicating the presence of the infecting agent in the culture obtained by inoculation with the samples of urine and kidney tissue, was found to be insufficient, as only 28 out of 45 cultures of E. coli isolated from the kidneys coincided with histologically confirmed cases of pyelonephritis.     

An experimental model of ascending pyelonephritis due toCandida albicans in rats

We developed a new experimental model of ascendingCandida pyelonephritis in female rats with leukopenia and vesicoureteral reflux. Rats were treated transperitoneally with cyclophosphamide (200 mg/kg) to induce leukopenia 3 days before and transurethrally with diluted acetic acid solution to induce vesicoureteral reflux 1 day before inoculation ofCandida albicans strain, ATCC 10259 (containing 10⁷ cells). Microscopy revealed acute pyelonephritis in whichCandida cells invaded from the fornix and/or papilla into the medulla within 3 days after inoculation. Between 7 and 28 days after inoculation, chronic pyelonephritis reached the cortex. The incidence of pyelonephritis increased gradually and was approximately 80% after 7 days.Candida colony counts of bladder urine specimens obtained by direct puncture were significantly greater in rats with pyelonephritis extending into the parenchyma than in those with pyelonephritis located along the pelvis (p<0.01). These results suggest that this rat model shows the characteristic feature of ascending pyelonephritis due toC. albicans and that the severity ofCandida pyelonephritis can be estimated fromCandida counts of bladder urine.     

A comparative study of the biological properties of pyelonephritogenic Escherichia coli isolated at different times in the infectious process

The comparative study of the biological properties of E. coli cultures, isolated from the urine of 7 patients two times during the first 11 days from the beginning of clinical manifestations of the exacerbation of chronic pyelonephritis, was conducted. In most cases the strains obtained as the result of the inoculations of the first and second urine samples belonged to the same serological and enzymatic variants. Still bacteria isolated in the second investigation, in contrast to E. coli obtained by the earlier inoculation of urine samples, often had no hemagglutinins and showed low adhesive capacity with respect to uroepithelium. Only in one out of 4 patient E. coli with antigen K1+ could be detected not only after the first inoculation, but also after the second one. In 4 patients E. coli cultures obtained as the result of the second isolation of these bacteria had lower content of sialic acid. Besides, differences in the sensitivity of E. coli strains isolated from the same patients in the course of the infectious process to the action of nonspecific protection factors of the body were established. The results obtained in the course of this study give more precise understanding of the existing conception of the pathogenesis of pyelonephritis.     

炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.     

Bone morphogenetic protein-7 expression in human pyelonephritis

Bone morphogenetic protein 7 (BMP- 7) is a member of the transforming growth factor (TGF) beta superfamily and is involved in regeneration, repair, and development of specific tissues, for example kidney and skeleton. The experimental studies have shown its protective role against fibrotic processes. Tubulointerstitial changes are present in the pyelonephritic kidney which progresses to fibrosis. Renal fibrosis may lead to the loss of renal function. The aim of this study was to investigate BMP-7 expression in acute and chronic pyelonephritis in humans. Seven patients with acute pyelonephritis and 7 with chronic pyelonephritis were treated in Department of Nephrology Clinical Hospital, Rijeka. Tissue biopsy was taken and renal tissue was studied histopathologically by use of hematoxylin and eosin and scored for diagnosis of pyelonephritis. BMP-7 expression was studied by immunohistochemical staining. BMP-7 expression was observed in the tubular area of the pyelonephritic kidneys. The expression of BMP- 7 was stronger in the acute pyelonephritic group and less in the chronic pyelonephritic group of patients. The results imply that BMP-7 has a role in chronic pyelonephritis. Tubular BMP-7 expression had a negative correlation with fibrosis and tubular, atrophy. Our results are suggesting that BMP- 7 plays an important protective role in renal inflammatory diseases preventing greater damage and fibrosis.     

A型肉毒毒素保护性抗原的基因修饰及高效表达

在A型肉毒毒素保护性抗原基因初步表达的基础上,为提高表达水平,依据EMBL的DNA数据库中A型肉毒毒素基因全序列,重新设计上游引物,通过修饰基因片段N端,保持氨基酸序列不变,从已获得的A型肉毒毒素与靶细胞起结合作用的重链C端基因中,扩增小的突变基因,克隆入pGEM-T载体进行测序,并以pBV220为表达载体构建重组表达质粒,在大肠杆菌中实现高效表达。结果表明,重组表达产物占全菌蛋白的40%,酶联检测重组表达产物具有特异结合活性。A型肉毒毒素保护性抗原基因的高效表达,为下一步基因工程抗毒素和疫苗的研制奠定了基础。     

An ultrastructural study of the renal medulla in experimental acute pyelonephritis.

Experimental acute pyelonephritis was produced in rats by a combination of intravenous administration of Escherichia coli, strain IMRU-54, and temporary unilateral mechanical ureteral obstruction. Structural alterations of the renal medulla were studied by light and electron microscopy. Major cellular alterations occurred in the vasa recta. Tubular and interstitial cells demonstrated minimal alterations after the brief period of acute inflammation. Polymorphonuclear leukocytes within tubular lumina contained structures resembling E. coli in nonprotoplasts-like form. Numerous protoplast-like organisms, to the exclusion of any other structural forms, were detected within the interstitium of the inner medulla. Nonprotoplast-like structures resembling E. coli were rarely observed in interstitium of the inner medulla. Following relief of ureteral obstruction, clearance of acute inflammation was rapid. In conclusion, hemoatogenous acute pyelonephritis induced by E. coli, IMRU-54, is able to inflict cytological and ultrastructural damage to structural elements of the inner and outer medulla of rats. Vasa recta incurred prominent alterations in endothelia and basement membranes, whereas tubular epithelia and interstitial cells had relatively good structural preservation. The data suggest that intravenously administered E. coli is capable to revert to a protoplast-like structure in the inner medulla.     

P2X1 receptor blockers reduce the number of circulating thrombocytes and the overall survival of urosepsis with haemolysin-producing Escherichia coli

Purinergic Signalling - Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially...     

Comparative characteristics of the adhesive properties of microorganisms isolated from the urine of children with chronic obstructive pyelonephritis

The adhesive properties of 215 cultures, including 215 Escherichia coli strains, 43 Klebsiella pneumoniae strains and 60 Pseudomonas aeruginosa strains isolated from the urine of 124 children with chronic obstructive pyelonephritis were studied in the direct hemagglutination test simultaneously with those of 30 E. coli strains and 20 K. pneumoniae strains isolated from the feces of 50 healthy children, as well as 60 P. aeruginosa strains isolated from children with parenteral infections of other localization. E. coli and K. pneumoniae strains isolated from the urine of children with chronic obstructive pyelonephritis were found to have D-mannose-resistant hemagglutinins (68% and 37.2%) and a combination of mannose-sensitive and mannose-resistant adhesins (44.6% and 13.3% respectively). P. aeruginosa strains isolated from the urine of urological patients in the postoperative period showed the presence of mannose-resistant hemagglutinins to a greater extent (76.6%) than those isolated from children with parenteral infections of other localization (45%).     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。