Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract.

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.

Repression of bacterial motility by a novel fimbrial gene product.

Proteus mirabilis is a common uropathogen in patients with long-term catheterization or with structural or functional abnormalities in the urinary tract. The mannose-resistant, Proteus-like (MR/P) fimbriae and flagellum are among virulence factors of P.mirabilis that contribute to its colonization in a murine model of ascending urinary tract infection. mrpJ, the last of nine genes of the mrp operon, encodes a 107 amino acid protein that contains a putative helix-turn-helix domain. Using transcriptional lacZ fusions integrated into the chromosome and mutagenesis studies, we demonstrate that MrpJ represses transcription of the flagellar regulon and thus reduces flagella synthesis when MR/P fimbriae are produced. The repression of flagella synthesis by MrpJ is confirmed by electron microscopy. However, a gel mobility shift assay indicates that MrpJ does not bind directly to the regulatory region of the flhDC operon. The isogenic mrpJ null mutant of wild-type P.mirabilis strain HI4320 is attenuated in the murine model. Our data also indicate that PapX encoded by a pap (pyelonephritis- associated pilus) operon of uropathogenic Escherichia coli is a functional homolog of MrpJ.

Role of Enterococcus faecalis surface protein Esp in the pathogenesis of ascending urinary tract infection.

Enterococcus faecalis bacteria isolated from patients with bacteremia, endocarditis, and urinary tract infections more frequently express the surface protein Esp than do fecal isolates. To assess the role of Esp in colonization and persistence of E. faecalis in an animal model of ascending urinary tract infection, we compared an Esp(+) strain of E. faecalis to its isogenic Esp-deficient mutant. Groups of CBA/J mice were challenged transurethrally with 10(8) CFU of either the parent or mutant strain, and bacteria in the urine, bladder, and kidneys were enumerated 5 days postinfection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the parent strain than from the bladder and urine of mice challenged with the mutant. Colonization of the kidney, however, was not significantly different between the parent and mutant strains. Histopathological evaluations of kidney and bladder tissue done at 5 days postinfection did not show marked histopathological changes consistent with inflammation, mucosal hyperplasia, or apoptosis, and there was no observable difference between the mice challenged with the parent and those challenged with the mutant. We conclude that, while Esp does not influence histopathological changes associated with acute urinary tract infections, it contributes to colonization and persistence of E. faecalis at this site.

The role of cytotoxic necrotizing factor-1 in colonization and tissue injury in a murine model of urinary tract infection.

Cytotoxic necrotizing factor-1 (CNF1) is commonly found in Escherichia coli isolates from patients with urinary tract infection (UTI). To determine whether CNF1 is an important UTI virulence factor we compared the ability of a clinical E. coli UTI isolate and a CNF1-negative mutant of that isolate to colonize and induce histological changes in the urinary tract in a murine model of ascending UTI. We found no evidence that the mutant strain was attenuated.

Serum immunoglobulin response and protection from homologous challenge by Proteus mirabilis in a mouse model of ascending urinary tract infection.

We tested the hypothesis that experimental Proteus mirabilis urinary tract infection in mice would protect against homologous bladder rechallenge. Despite production of serum immunoglobulin G (IgG) and IgM (median titers of 1:320 and 1:80, respectively), vaccinated (infected and antibiotic-cured) mice did not show a decrease in mortality upon rechallenge; the survivors experienced only modest protection from infection (mean log(10) number of CFU of P. mirabilis Nal(r) HI4320 per milliliter or gram in vaccinated mice versus sham-vaccinated mice: urine, 100-fold less [3.5 versus 5.5; P = 0.13]; bladder, 100-fold less [3.1 versus 5.1; P = 0.066]; kidneys, 40-fold less [2.7 versus 4.3; P = 0.016]). Western blots using protein from the wild-type strain and isogenic mutants demonstrated antibody responses to MR/P and PMF fimbriae and flagella. There was no correlation between serum IgG or IgM levels and protection from mortality or infection. There was a trend toward elevated serum IgA titers and protection from subsequent challenge (P >/= 0.09), although only a few mice developed significant serum IgA levels. We conclude that prior infection with P. mirabilis does not protect significantly against homologous challenge.

ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells.

The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.

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.

Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis.

Proteus mirabilis fimbriae: construction of an isogenic pmfA mutant and analysis of virulence in a CBA mouse model of ascending urinary tract infection.

Proteus mirabilis, a cause of urinary tract infection and acute pyelonephritis, produces a number of different fimbriae. An isogenic fimbrial mutant of P. mirabilis HI4320 was constructed by marker exchange with delta pmfA::aphA to determine the role of the P. mirabilis fimbriae (PMF) in hemagglutination and in virulence in the CBA mouse model of ascending urinary tract infection. The pmfA mutant, which did not express the 19,500-Da major subunit of PMF, colonized the bladders of transurethrally challenged CBA mice (n = 20 in each group) in numbers 83-fold lower than those of the wild-type strain (mutant, log10 4.87 CFU/g; wild-type strain, log10 6.79 CFU/g; P = 0.023). However, the mutant colonized the kidneys in numbers similar to those of the wild-type strain. Hemagglutination patterns of the mutant ruled out the involvement of PMF in both mannose-resistant, Proteus-like and mannose-resistant, Klebsiella-like hemagglutination. Similarly, PMF does not appear to be involved in adherence to uroepithelial cells (UEC), since the mutant was as adherent as the wild-type strain (mutant, 14.1 +/- 11.7 mean bacteria per UEC, 60% of UEC with > or = 10 bacteria; wild-type strain, 18.1 +/- 16.2 mean bacteria per UEC, 68% of UEC with > or = 10 bacteria; not significantly different). These data suggest a role for PMF in colonization of the bladder but not in colonization of kidney tissue. PMF appear not to be responsible for mannose-resistant, Proteus-like or mannose-resistant, Klebsiella-like hemagglutination.

Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of alpha Gal(1-4) beta Gal binding in virulence of a wild-type strain.

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.

Urethral obstruction of 6 hours or less causes bacteriuria, bacteremia, and pyelonephritis in mice challenged with "nonuropathogenic" Escherichia coli.

Urethral obstruction may be caused by prostatic hypertrophy, urethral stricture, or encrustation of a urethral-catheter lumen. Bacteriuria often complicates these obstructions. The sequelae include fever, acute pyelonephritis, chronic renal inflammation, and death. We hypothesized that even brief obstruction of the urinary tract containing a nonvirulent bacterium would result in these complications. Mice challenged transurethrally with Escherichia coli FN414, which is rapidly eliminated from normal mice without causing bacteriuria, bacteremia, or renal pathology, were subjected to reversible urethral obstruction by coating the urethral meatus with collodion for 1, 3, or 6 h. The majority of mice obstructed for 1 h demonstrated parenchymal renal inflammation 48 h later. At the end of 3 h of obstruction, 9 of 10 mice were bacteremic; some bacteremias were present at 48 h after removal of the obstruction. At that time, more severe renal inflammation was seen in these mice. As little as 6 h of obstruction resulted not only in the acute changes described above but also in chronic renal inflammation and fibrosis in the majority of animals sacrificed 3 and 6 weeks later. Additional studies demonstrated that urethral obstruction enhanced the uropathogenicity of another nonpathogenic E. coli strain (K-12 strain HB101) and caused more severe renal lesions in mice challenged with E. coli CFT073, isolated from a patient with symptoms of pyelonephritis. These findings demonstrate that brief urethral obstruction may (i) induce organisms which are cleared rapidly from the normal urinary tract to cause bacteriuria, bacteremia, and pyelonephritis and (ii) intensify the renal lesions caused by a uropathogen.

Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a significant cause of bacteriuria and acute pyelonephritis in humans, produces urease. This high-molecular-weight, multimeric, cytoplasmic enzyme hydrolyzes urea to ammonia and carbon dioxide. To assess the role of urease in colonization, urolithiasis, and acute pyelonephritis in an animal model of ascending urinary tract infection, we compared a uropathogenic strain of P. mirabilis with its isogenic urease-negative mutant, containing an insertion mutation within ureC, the gene encoding the large subunit of the enzyme. Mice challenged transurethrally with the parent strain developed significant bacteriuria and urinary stones. The urease-negative mutant had a 50% infective dose of 2.7 x 10(9) CFU, a value more than 1,000-fold greater than that of the parent strain (2.2 x 10(6) CFU). The urease-positive parent strain reached significantly higher concentrations and persisted significantly longer in the bladder and kidney than did the mutant. Indeed, in the kidney, the parent strain increased in concentration while the mutant concentration fell so that, by 1 week, the parent strain concentration was 10(6) times that of the mutant. Similarly, the urease-positive parent produced significantly more severe renal pathology than the mutant. The initial abnormalities were in and around the pelvis and consisted of acute inflammation and epithelial necrosis. By 1 week, pyelitis was more severe, crystals were seen in the pelvis, and acute pyelonephritis, with acute interstitial inflammation, tubular epithelial cell necrosis, and in some cases abscesses, had developed. By 2 weeks, more animals had renal abscesses and radial bands of fibrosis. We conclude that the urease of P. mirabilis is a critical virulence determinant for colonization, urolithiasis, and severe acute pyelonephritis.

Mouse models of short- and long-term foreign body in the urinary bladder: analogies to the bladder segment of urinary catheters.

Catheter-associated bacteriuria is the most common infection occurring in hospitals, where urethral catheters are generally in place for a few days, and in nursing homes, where catheters may be in place for months or years. We developed murine models with intrabladder urinary catheters for studying complications of bacteriuria in short- and long-term catheterization. In the short-term model, a catheter segment was inserted transurethrally and lay free within the bladder lumen. Half of the animals expelled segments during a 2-to-7-day period, durations similar to catheterizations in hospitalized patients. For studies of long-term catheter use, the catheter segment was secured within the bladder by a single suture for up to 12 months. Antibiotics administered for 7 days after catheter placement and housing mice in cages with wire screen floors reduced spontaneous bacteriuria to an acceptably low incidence rate of only 7%. Proteus mirabilis bacteriuria of high concentration provoked the same complications that are common in patients with long-term catheters: acute pyelonephritis, chronic renal inflammation, and struvite stone formation. These models allow inoculation of the bacteria of interest and are suitable for studies of short- and long-term foreign body-associated bacteriuria and its complications.

Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains.

Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains (greater than or equal to 70% of mice challenged developed greater than or equal to 10(3) CFU/g of kidney after 48 h) were selected for study. Each strain displayed mannose-resistant hemagglutination of human O erythrocytes; three strains were phenotypically and genotypically hemolytic. Pyelonephritogenic strains were significantly more cytotoxic (30.1 +/- 9.5% LDH release after 18 h) than eight fecal control strains (13.5 +/- 11.5% LDH release; P = 0.0068). We selected the most cytotoxic strain, CFT073, for further study. Sterile filtrate from this hemolytic strain was significantly more cytotoxic than was the filtrate of the fecal control strain, FN414. Transposon mutagenesis of CFT073 with TnphoA abolished hemolytic activity and cytotoxicity by both whole cells and sterile filtrate. Southern blot analysis revealed that the Tnphoa insertion mapped to the E. coli chromosomal hly determinant within a 12-kilobase SalI restriction fragment. Transformation of a nonhemolytic strain, CPZ005 with plasmid pSF4000, which carries a cloned hemolysin determinant, resulted in highly elevated cytotoxicity. Light micrographs of proximal tubular epithelial cell cultures demonstrated cell damage by pyelonephritogenic strains that was not induced by a fecal strain or the hemolysin-deficient mutant. Results indicate that pyelonephritogenic E. coli strains are more frequently cytotoxic for a putative target, that is, human renal tubular epithelium, than are fecal isolates. Hemolysin, in some strains, is apparently responsible for this cytotoxicity.

Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a urease-producing uropathogen, causes serious urinary tract infections in humans. To specifically evaluate the contribution of urease to virulence, a mutation was introduced into P. mirabilis HI4320 by homologous recombination. Virulence was assessed in the CBA mouse model of ascending urinary tract infection. Twenty mice each were challenged transurethrally with P. mirabilis HI4320 and its urease-negative derivative (1 x 10(9) to 2 x 10(9) CFU). At 48 h animals were sacrificed and the mean log10 CFU per milliliter of urine (parent, 6.23; mutant, 4.19; P = 0.0014) or per gram of bladder (parent, 6.29; mutant, 4.28; P = 0.0002), left kidney (parent, 4.11; mutant, 1.02; P = 0.00009), and right kidney (parent, 4.11; mutant, 2.43; P = 0.036) were all shown to be significantly different. These data demonstrate a role for urease as a critical virulence determinant for uropathogenic P. mirabilis.

Uropathogenicity in rats and mice of Providencia stuartii from long-term catheterized patients.

Providencia stuartii, a frequent and persistent isolate from the urinary tract of chronically catheterized elderly patients, is multiply antibiotic resistant and may cause fatal bacteremia in those patients. We studied P. stuartii strains in rats and mice to determine differences in uropathogenicity. Strains studied varied in expression of factors which contribute to pathogenicity of other bacterial species. Urinary tract responses following challenge with P. stuartii strain HO (factors expressed) were similar to responses reported for uropathogenic E. coli strains both in bacterial persistence and histologic change. In animals similarly challenged with P. stuartii strain RO (factors not expressed), responses were similar to those reported for non-uropathogenic E. coli strains. Results indicate that: a) animal model studies may be useful in differentiating P. stuartii strains based on uropathogenicity, b) P. stuartii uropathogenicity may be related to identifiable factors associated with virulence in other species, and c) the CBA mouse model appears to be the most suitable for studies of P. stuartii uropathogenicity.