Vergleichende Untersuchungen über die hämatogene und die ascendierende Pyelonephritis bei Ratten

Zusammenfassung Bei insgesamt 104 Ratten wurde mit E. coli oder Proteus eine Pyelonephritis erzeugt. Die Keime wurden intravenös oder in die Harnblase appliziert. Durch vorangegangene transabdominelle Nierenmassage, Einlegen einer Perle in die Harnblase oder einseitige temporäre Ureterligatur konnte die Entstehung der Pyelonephritis begünstigt werden. Histologische und bakteriologische Untersuchungen der Nieren erfolgten nach 24 Std bis 8 Wochen.Allein aufgrund der histologischen Veränderungen ließ sich nicht erkennen, ob die Pyelonephritis auf hämatogenem oder ascendierendem Weg entstanden ist, da keine morphologischen Unterschiede auftraten. Die hier gewählten Versuchsanordnungen, bakteriologische Untersuchungen von Blut und Nierengewebe und theoretische Überlegungen sprechen für die hämatogene Genese der Pyelonephritis in jedem unserer Versuchsmodelle.Proteus ruft schwerere Entzündungen in den Nieren hervor als E. coli. Von den Co-Faktoren war die Ureterligatur am wirksamsten. Sämtliche hier angewandten Versuchsanordnungen scheinen geeignet, eine experimentelle Pyelonephritis bei Ratten mit Erfolg zu erzeugen.

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Comparative studies on hematogenous and ascending pyelonephritis in rats

Summary Experimental pyelonephritis was produced in 104 rats with E. coli and proteus which were administered intravenously or into the urinary bladder. The development of pyelonephiritis was favoured by preceeding transabdominal massage of the kidneys, by implantation of a pearl into the urinary bladder, or by temporary, unilateral ligation of the ureter. Histologic and bacteriologic examinations were made 24 hours to 8 weeks afterwards.It was impossible to decide merely from the histologic lesion whether pyelonephritis was of hematogenous or ascending origin, because no morphologic differences developed. The experimental procedure chosen, the bacteriological examinations of blood and kidney tissue, and the theoretical considerations favour the theory of hematogenous genesis of pyelonephritis in all of our experiments.Inflammation of the kidneys caused by proteus is more severe than that caused by E. coli. Ureteral ligation was the most effective of the co-factors. All of the experimental procedures used seem to be suitable for causing experimental pyelonephritis in rats.

     

Immunization Against Retrograde Pyelonephritis: I. Production of an Experimental Model of Severe Ascending Escherichia coli Pyelonephritis Without Bacteremia in Rats

Retrograde pyelonephritis was produced in rats by introducing into the bladder a small refluxing inoculum of Escherichia coli that would enter the renal pelvis but not the blood stream. At the same time the left ureter was partially obstructed for 18 hours. This model differs from previous attempts to produce E coli pyelonephritis with large (0.6 ml) volumes infused into the bladder, because such large volumes cause bacteremia and hematogeneous pyelonephritis. Since retrograde E coli pyelonephritis in patients is not accompanied by positive blood cultures, the model described in this report is believed to accurately mimic human pyelonephritis and to allow a realistic approach to the study of immunity against retrograde infection in the urinary tract.     

Bacteremia in the Pathogenesis of Retrograde E coli Pyelonephritis in the Rat

Female rats deprived of water overnight, and then given 1.0 ml of E coli 0111:B4 via the urethra, developed pyelonephritis. A nearly absolute association was found between the occurrence of bacteremia after the transurethral infusion and the development of pyelonephritis. An identical lesion was produced by a combination of forniceal damage and intravenous injection of E coli. The kidney damaged by reflux was shown to be more susceptible to hematogenous pyelonephritis than the obstructed kidney and the distribution of the infection was due to localization of bacteria in the damaged fornix but not to the route of infection. The induction of retrograde E coli pyelonephritis in the rat required a tear in the pelvic epithelium creating pyelovenous communications, and the resultant bacteremia produced pyelonephritis. The incidence of ureteral reflux and the volume of inoculum that refluxed to the renal pelvis was shown radiologically to be a function of bladder distensibility, which is reduced by withholding water for a few hours. In this system, retrograde E coli pyelonephritis developed from a combination of two factors: (1) reflux-induced damage to the renal pelvis so that E coli are introduced into the kidney and (2) hematogenous infection of the damaged kidney.     

Immunization Against Retrograde Pyelonephritis: II. Prevention of Retrograde Escherichia coli Pyelonephritis with Vaccines

Vaccination with heat-killed or formalinized cells of E coli 0:111, or E coli 06 (Williams), prevented retrograde E coli pyelonephritis. Since there was no bacteremia and no urinary antibody, the vaccination appeared to protect by immune reactions operating in the kidney itself. The vaccine failed to protect against a highly virulent form of E coli 06 (Riffle), possibly because the amount of antibody to its lipopolysaccharide was inadequate. Since all three strains possessed K antigen in approximately equal amounts, the difference in results was not attributed to its presence.     

An experimental model for ascending acute pyelonephritis caused by Escherichia coli or proteus in rats.

Experimental, ascending acute pyelonephritis in rats was produced by injecting 0 x 5 ml of 10(9) bacteria/ml into the urinary bladder via the urethra. No traumatic manipulation of the ureters of kidneys was necessary. A grading system for kidney lesions based on macro- and microscopical examination was used. The capacity of different Escherichia coli and proteus strains to induce acute pyelonephritis was tested, and the E. coli 06K13H1 strain and the Proteus mirabilis 03H1 strain were especially capable of causing urinary tract infection. For the P. mirabilis 03H1 strain, a dominance of right kidney lesions was noted in contrast to the E. coli 06K13H1 strain which did not show any side preference.     

Antiviral activity of Brucella abortus preparations; separation of active components.

Injection into mice of heat-killed Brucella abortus or aqueous ether-extracted B. abortus (Bru-pel) induced a "virus-type" interferon response, with peak titers at 6.5 h. The animals also were protected against challenge with otherwise lethal doses of Semliki forest virus. Extraction of either heated B. abortus or BRU-PEL with a mixture of chloroform-methanol (2:1, vol/vol) (C-M( yielded an insoluble residue (extracted cells) and a C-M extract. Neither extracted cells nor C-M extract alone induced interferon or afforded protection against Semliki forest virus infection in mice. Full interferon-inducing and protective activity was restored when extracted cells were recombined with C-M extract. C-M extract from heat-killed Escherichia coli also was effective in restoring activity to extracted Brucella cells. Neither heat-killed E. coli nor its C-M extract was active, nor was C-M estracted E. coli recombined with the C-M extract from B. abortus. These results suggest that the interferon-inducing and antiviral protective properties of B. abortus are constituted of a C-M-extractable component that is common to B. abortus and E. coli and an unextractable component that is unique to B. abortus.     

Protection Against Acute, Ascending Pyelonephritis Caused by Escherichia coli in Rats, Using Isolated Capsular Antigen Conjugated to Bovine Serum Albumin

We found that isolated Escherichia coli K13 antigen conjugated to bovine serum albumin, in contrast to isolated, non-conjugated K13, was highly immunogenic and induced protection against acute pyelonephritis caused by E. coli O6K13H1 in rats.     

The IrgA homologue adhesin Iha is an Escherichia coli virulence factor in murine urinary tract infection

The role of the Escherichia coli iron-regulated gene homologue adhesin (Iha) in the pathogenesis of urinary tract infections (UTIs) is unknown. We performed a series of complementary analyses to confirm or refute the hypothesis that Iha is a virulence factor in uropathogenic E. coli. Fecal E. coli isolates exhibited significantly lower prevalences of iha (range, 14 to 22%) than did clinical isolates from cases of pediatric cystitis or pyelonephritis, adult pyelonephritis or urosepsis, or bacteremia (range, 38 to 74%). Recombinant Iha from E. coli pyelonephritis isolate CFT073 conferred upon nonadherent E. coli ORN172 the ability to adhere to cultured T-24 human uroepithelial cells. In a well-established mouse model of ascending UTI, CFT073 and its derivative UPEC76 (a pap [P fimbriae] mutant version of strain CFT073) each significantly outcompeted their respective iha deletion mutants in CBA/J mice 48 h after bladder challenge (P < 0.03 for urine, both kidneys, and bladders of both constructs, except for bladders of mice challenged with UPEC76 and its deletion mutant, where P = 0.11). These data suggest that Iha(CFT073) is a virulence factor and might be a target for anti-UTI interventions.     

Comparison of Escherichia coli Strains Recovered from Human Cystitis and Pyelonephritis Infections in Transurethrally Challenged Mice

Urinary tract infection, most frequently caused by Escherichia coli, is one of the most common bacterial infections in humans. A vast amount of literature regarding the mechanisms through which E. coli induces pyelonephritis has accumulated. Although cystitis accounts for 95% of visits to physicians for symptoms of urinary tract infections, few in vivo studies have investigated possible differences between E. coli recovered from patients with clinical symptoms of cystitis and that from patients with symptoms of pyelonephritis. Epidemiological studies indicate that cystitis-associated strains appear to differ from pyelonephritis-associated strains in elaboration of some putative virulence factors. With transurethrally challenged mice we studied possible differences using three each of the most virulent pyelonephritis and cystitis E. coli strains in our collection. The results indicate that cystitis strains colonize the bladder more rapidly than do pyelonephritis strains, while the rates of kidney colonization are similar. Cystitis strains colonize the bladder in higher numbers, induce more pronounced histologic changes in the bladder, and are more rapidly eliminated from the mouse urinary tract than pyelonephritis strains. These results provide evidence that cystitis strains differ from pyelonephritis strains in this model, that this model is useful for the study of the uropathogenicity of cystitis strains, and that it would be unwise to use pyelonephritis strains to study putative virulence factors important in the development of cystitis.     

An experimental model of ascending pyelonephritis in the rabbit

Summary A model of acute ascending purulent pyelonephritis was produced in rabbits by injection of an E. coli suspension into the urinary bladder with subsequent squeezing of the bladder wall. An important factor influencing the penetration of bacteria from the bladder into the renal pelvis was a sufficient volume of the bacterial suspension. Penetration of bacteria into the renal parenchyma and their multiplication in the renal tissue was rendered possible by temporary ligature of the ureter prior to the injection of bacterial suspension.

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Experimentelles Modell einer ascendierenden Pyelonephritis beim Kaninchen

Zusammenfassung Das Modell einer akuten ascendierenden eitrigen Pyelonephritis wurde bei Kaninchen mittels Injektion einer E. coli-Suspension in die Harnblase und nachfolgender Kompression der Blasenwand erzeugt. Ein genügend großes Volumen der Keimsuspension ist ein wichtiger Faktor, um das Eindringen der Keime aus der Blase in das Nierenbecken zu bewirken. Das Eindringen der Keime in das Nierenparenchym und ihre Vermehrung im Nierengewebe wird durch eine temporäre Harnleiterligatur vor der Keiminjektion in die Blase ermöglicht.

     

Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin.

A model for ascending unobstructed urinary tract infection was developed in mice to study the pathogenesis of urinary tract infection induced by Escherichia coli associated with urinary tract infection in humans. Specifically, the model was designed to monitor the initial stages of the infectious process, e.g., bacterial adhesion. Mice were selected since the specificity and intensity of bacterial attachment of pyelonephritogenic E. coli strains to human and mouse uroepithelial cells were similar. Female mice were infected by urethral catheterization and installation of bacteria in the urinary bladder. To maximize clearance of unattached bacteria, no obstructive manipulations were performed. After sacrifice, the persistence of bacteria in kidneys and bladder was determined by viable counts on homogenized tissues. The experimental infection was standardized by using one pyelonephritis (HU734) and one normal fecal (414) E. coli isolate. With both strains all of the bladders became infected, but E. coli 414 was eliminated more rapidly than HU734. The percentage of positive kidney cultures increased with the bacterial inoculum concentration and volume. An inoculum of 0.05 ml containing 10(10) bacteria per ml was selected, giving the highest percentage of positive kidney cultures without detectable bacterial spread to the blood stream. The variation in the percentage of positive kidney cultures possibly depended on the degree of vesicoureteric reflux in the individual animals. Both in the kidneys and in the urinary bladders, strain HU734 yielded higher numbers of bacteria at 24 h and persisted longer than did strain 414. Several E. coli pyelonephritis isolates with properties associated with virulence in the human urinary tract consistently were recovered from mouse kidneys and bladders in higher numbers than E. coli strains of human fecal origin lacking those properties. The role of bacterial adhesion per se is the topic of the accompanying paper.     

Effect of Escherichia coli and Lactobacillus rhamnosus on macrophage inflammatory protein 3 alpha, tumor necrosis factor alpha, and transforming growth factor beta release by polarized rat uterine epithelial cells in culture

Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria. Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection. UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER). Polarized epithelial cell cultures were treated apically with live or heat-killed Escherichia coli or Lactobacillus rhamnosus prior to collection of basolateral media after 24 h of incubation. Coculture of polarized UECs with live E. coli had no effect on epithelial cell TER. In response to exposure to live E. coli, epithelial cell basolateral release of macrophage inflammatory protein 3 alpha (MIP3 alpha) and tumor necrosis factor alpha (TNF-alpha) increased at a time when basolateral release of biologically active transforming growth factor beta (TGF-beta) decreased. Incubation of UECs with heat-killed E. coli resulted in an increased basolateral release of MIP3 alpha and TNF-alpha, without affecting TER or TGF-beta. In contrast to E. coli, live or heat-killed L. rhamnosus had no effect on TER or cytokine release. These studies indicate that polarized rat UECs respond to gram-negative E. coli by releasing the cytokines MIP3 alpha and TNF-alpha, signals important to both the innate and adaptive immune systems. These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract.     

Inverse relationship between severity of experimental pyelonephritis and nitric oxide production in C3H/HeJ mice

The contribution of nitric oxide to host resistance to experimental pyelonephritis is not well understood. We examined whether the inhibition of nitric oxide synthesis alters the sensitivity of lipopolysaccharide (LPS) responder (C3H/HeN) and nonresponder (C3H/HeJ) mice to experimental Escherichia coli pyelonephritis. C3H/HeJ and C3H/HeN mice were implanted subcutaneously with minipumps containing an inhibitor of nitric oxide, NG-nitro-L-arginine methyl ester (L-NAME), or a corresponding vehicle. Ascending urinary tract infection by bladder catheterization with two strains of E. coli, an O75 strain bearing Dr fimbriae and an O75 strain bearing P fimbriae, was developed in tested animals. Twenty-four hours following bladder infection, the kidneys of C3H/HeN and C3H/HeJ mice were colonized at a similar rate. However, 5 weeks postinoculation, C3H/HeN mice cleared infection while C3H/HeJ mice showed persistent colonization. Twenty-four hours following infection, C3H/HeN mice treated with L-NAME showed no significant increase of renal tissue infection compared to the saline-treated control group. However, L-NAME-treated C3H/HeJ mice showed an approximately 100-fold increase in E. coli infection rate compared to the saline-treated controls in the Dr+ group but showed no change compared to those in the P+ group. Dissemination of Dr+ E. coli but not P+ E. coli to the liver and uterus was significantly enhanced with L-NAME treatment in C3H/HeJ mice only. Nitric oxide had no direct killing effect on E. coli in vitro. Nitrite production by various organs was found to be significantly lower in C3H/HeJ mice than in C3H/HeN mice. Alteration of nitric oxide and LPS responsiveness was significantly associated with the increased sensitivity of C3H/HeJ mice to experimental Dr+ but not to P+ E. coli pyelonephritis. These findings are consistent with the hypothesis that nitric oxide synthase activity in concert with LPS responsiveness may participate in the antibacterial defense mechanisms of the C3H mouse urinary tract. This phenomenon is strain dependent and possibly related to the invasive properties of E. coli.     

Enhanced siderophore production and mouse kidney pathogenicity in Escherichia coli grown in urine.

Fifteen siderophore producing urinary isolates of Escherichia coli were compared for aerobactin and enterochelin production in trypticase soy broth and pooled normal human urine. Significant increase in siderophore production (both phenolate and hydroxamate) was observed when organisms were grown in urine. Mouse kidney pathogenic potential of the strains grown in urine was compared with that of bacteria grown in trypticase soy broth in an ascending model of pyelonephritis in female Swiss Webster mice. Organisms grown in urine and instilled into a mouse bladder demonstrated markedly enhanced renal pathogenicity (p less than 0.01). Further information about the influence of urinary constituents on siderophore production could help in understanding the pathogenesis of pyelonephritis.     

Protection against ascending Escherichia coli pyelonephritis in rats and significance of local immunity.

Acute pyelonephritis in rats caused by Escherichia coli O6K13H1 produced serum and urinary antibodies against O6 and K13 antigens. This was also seen after intravesical immunization with Formalin-killed bacteria. Both intraperitoneal and intravesical immunization with these Formalin-killed bacteria protected against ascending urinary tract infection induced by homologous bacteria. Passive transfer of urine containing both O6 and K13 antibodies also protected against infection. By absorption experiments it was shown that K13 antibodies were especially important.     

DNA sequences of three papA genes from uropathogenic Escherichia coli strains: evidence of structural and serological conservation.

Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Escherichia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specific binding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tip proteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and has been employed in vaccines despite its lack of Gal alpha(1-4)Gal receptor specificity. Animal recipients of PapA pilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologous Gal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were determined for three E. coli clones expressing F7(1), F7(2), and F9 pili and were compared with corresponding sequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbent assays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F7(1), F7(2), F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate the concept that papA genes are highly homologous and encode proteins which exhibit greater than 70% homology among pili of different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions and constitute the basis for serological diversity of these pili. Although there are differences in primary structures among these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Gal pili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted strongly or moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there are common immunogenic domains among these proteins. These additional data further support the hypothesis that broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might be developed.     

Experimental Gestational Pyelonephritis Induces Preterm Births and Low Birth Weights in C3H/HeJ Mice

Urinary tract infections (UTIs) are associated with approximately 27% of premature births. Escherichia coli is the most frequent causal agent of UTIs and expresses virulence factors, including surface adhesins that recognize specific host tissue receptors. We have reported that E. coli Dr adhesin recognizes decay-accelerating factor as the host tissue receptor and that these receptors are increased during pregnancy. Induction of pathogenesis is a cumulative effect of the host-pathogen relationship involving specific host factors and virulence characteristics of the invading organism. Recently, an experimental model of chronic pyelonephritis has been developed with E. coli bearing Dr adhesin (E. coli Dr(+)) in nonpregnant lipopolysaccharide hyporesponder C3H/HeJ mice. In this study, we investigated the role of E. coli Dr(+) on the outcome of pregnancy in C3H/HeJ mice. Groups of pregnant mice were infected with E. coli Dr(+) or its isogenic mutant which does not bear the Dr adhesin (E. coli Dr(-)) by urethral catheterization. Nearly 90% of pregnant mice infected with E. coli Dr(+) delivered preterm (before 90% gestation) compared to 10% of mice infected with E. coli Dr(-) and none of the mice treated with phosphate-buffered saline (PBS). Also, there was a significant reduction in fetal birth weight in the E. coli Dr(+)-infected group compared to the E. coli Dr(-)- and PBS-treated groups (P = 0.003). This experimental model of E. coli Dr(+)-induced preterm delivery in mice may help in understanding the molecular mechanisms involved in UTI-induced preterm labor involving bacterial adhesins.     

Identification of Escherichia coli genes associated with urinary tract infections

Escherichia coli is the most common cause of urinary tract infections (UTIs). E. coli genes epidemiologically associated with UTIs are potentially valuable in developing strategies for treating and/or preventing such infections as well as differentiating uropathogenic E. coli from nonuropathogenic E. coli. To identify E. coli genes associated with UTIs in humans, we combined microarray-based and PCR-based analyses to investigate different E. coli source groups derived from feces of healthy humans and from patients with cystitis, pyelonephritis, or urosepsis. The cjrABC-senB gene cluster, sivH, sisA, sisB, eco274, and fbpB, were identified to be associated with UTIs. Of these, cjrABC-senB, sisA, sisB, and fbpB are known to be involved in urovirulence in the mouse model of ascending UTI. Our results provide evidence to support their roles as urovirulence factors in human UTIs. In addition, the newly identified UTI-associated genes were mainly found in members of phylogenetic groups B2 and/or D.     

Experimental Escherichia coli ascending pyelonephritis in rats: active peroral immunization with live Escherichia coli.

Peroral immunization with a live strain of Escherichia coli O6K13H1 against experimental ascending pyelonephritis caused by the same strain was studied in rats, and the effect of immunization on antibody titers against the O and K antigens and lipid A was determined. Peroral immunization with live bacteria protected significantly against pyelonephritis. Sera collected 1 week after infection from the immunized group were increased in immunoglobulin G (IgG) anti-O6 and IgM anti-K13 in comparison with the nonimmunized group. The peroral immunization did not correspondingly affect the response to lipid A. In urine, there was an IgG antibody response to the O6 antigen. In bronchopulmonary secretion, IgM, IgG, and IgA antibodies to O6 were detected. Perorally immunized animals had significantly higher levels of IgG and IgA anti-O6 compared with the nonimmunized group 1 week after infection. Passive transfer of anti-lipid A did not increase resistance against pyelonephritis.     

EXPERIMENTAL PYELONEPHRITIS IN MICE FOLLOWING ASCENDING INFECTION WITH E. COLI

The initial interaction between uroepithelial cells and Escherichia coli which has adhesive or invasive activity for cultured cells was studied ultrastructurally at the in situ site of infection in the model of ascending pyelonephritis in mice. The densely piliated adhesive strain E77156 isolated from the urine of a patient with urinary tract infection adhered to the pelvic and renal tubular epithelial cells and colonized on their cell surfaces and thereafter in the cytoplasm. The non-piliated invasive strain 633–65 isolated from a patient with dysentery-like syndrome did not colonize on the uroepithelial cell surfaces but easily penetrated into the cytoplasm of these cells. Thereafter multiplication was observed in their cytoplasm. Neither strain scarcely penetrated into the interstitium via the basement membrane of the renal tubules.