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While Proteus mirabilis causes less than 10% of uncomplicated urinary tract infections (UTIs), it is much more frequently isolated from patients with complicated UTIs, i.e., those with functional or anatomic abnormalities of or with chronic foreign bodies in the urinary tract (17, 21, 29). For example, in those with long-term catheters in place, nearly half of urine specimens contain P. mirabilis at concentrations of 10⁵ CFU/ml (24). This bacterium causes not only cystitis and acute pyelonephritis (5-7, 23) but also urinary stones, a result of expression of a highly active urease. Stone formation, a hallmark of infection with this organism, adds another dimension to the already complicated urinary tract (8, 18, 19).

Prevention of P. mirabilis UTIs is clearly a worthy goal, and thus, the concept of a vaccine has been pursued (15, 20). A vaccine against this organism may be feasible for several reasons. First, the species is quite homogeneous with respect to expression of surface antigens (14). Second, P. mirabilis is present in the fecal flora of <5% of individuals (25) and, thus, preventing its colonization of the host should not result in disruption of normal bowel flora. Finally, patient populations that would benefit from such a vaccine are well defined and include those with known anatomically or functionally abnormal urinary tracts, possibly women with recurrent UTIs, and those early in the course of long-term catheterization. As a first step toward the development of a vaccine, we assessed antibody response to whole bacteria and specific antigens and immunity to homologous reinfection in mice that had been inoculated transurethrally with a virulent P. mirabilis strain and subsequently cured by antibiotic treatment.

Experimental infection (vaccination). Live P. mirabilis HI4320, a strain recovered from the urine of a patient with catheter-associated bacteriuria and a mouse uropathogen (11), was used to assess immunity following transurethral challenge (vaccination). A nalidixic acid-resistant mutant of P. mirabilis HI4320 (P. mirabilis Nal^r HI4320; nalidixic acid MIC of 512 µg/ml) was used to challenge mice 5 weeks later (challenge). For mouse vaccination and challenge, P. mirabilis was grown overnight on Trypticase soy agar (TSA) (BBL, Cockeysville, Md.). Bacteria were harvested into phosphate-buffered 0.9% sodium chloride, pH 7.2 (PBS; BBL), and adjusted to approximately 2 × 10⁸ CFU/ml for P. mirabilis HI4320 and approximately 2 × 10⁷ CFU/ml for P. mirabilis Nal^r HI4320, using McFarland turbidity standards confirmed by spread plate enumeration (Spiral Systems, Bethesda, Md.). On day 1, mice were divided into vaccination (60 mice) and sham vaccination (30 mice) groups (Fig. 1). Vaccination group mice were challenged by the transurethral route using a previously described procedure (10). Sham-vaccinated mice were similarly infused with 50 µl of PBS. The catheter was removed immediately after transurethral infusion, and mice were returned to their cages and cared for by the normal routine. As described previously (10), in each experiment, one mouse was used to assess whether the inoculum refluxed into the kidney during the challenge procedure. Vaccinated and sham-vaccinated mice were observed daily for 4 weeks. During the observation period, sick and moribund mice were sacrificed by exposure to an overdose of CO₂. On days 28 to 31, ampicillin (500 mg/ml) was added to the mouse drinking water daily to eradicate residual P. mirabilis from the urinary tract. On day 32, tap water use was restored and mice were held for an additional 3 days to allow washout of the ampicillin. On day 35, urine samples were collected from all of the mice and cultured.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Flow chart of transurethral vaccination and challenge of mice with P. mirabilis.

Homologous challenge. Thirty mice in each of the vaccinated and sham-vaccinated groups were challenged transurethrally with 10⁶ CFU of P. mirabilis Nal^r HI4320 as described above. An additional 10 vaccinated mice were challenged only with 50 µl of PBS (sham challenge). Mice were examined daily and sacrificed 7 days after challenge (day 42) by using an overdose of CO₂. At sacrifice, the abdomen was opened aseptically by a midline incision and urine was aspirated from the bladder with a tuberculin syringe for quantitative bacteriologic culture. Then, after tying of the proximal end of each ureter, the bladder was washed by injecting and aspirating sterile saline. The bladder and kidneys were removed aseptically: the bladder and one half of each kidney were separately homogenized in PBS using a sterile glass grinder (Kontes, Inc., Vineland, N.J.). Urine and the homogenized tissues were quantitatively cultured on TSA and TSA containing nalidixic acid at 50 µg/ml by the spread plate technique. The mean number of CFU per milliliter of urine or gram of bladder or kidney was determined after 24 h of incubation at 37°C. Nalidixic acid-containing TSA was used to distinguish the nalidixic acid-resistant challenge strain from any residual nalidixic acid-susceptible strains of the original infection (i.e., the vaccination).

Statistical methods. Mean numbers of CFU per milliliter or gram from cultures of urine or tissue homogenates and mean histologic scores were compared by Student's t test. Differences in the numbers of mice with the bladder or kidneys colonized by the challenge organism were compared by chi-square analysis. The correlation between immunoglobulin response and quantitative infection in each mouse was measured by Spearman's rank order correlation coefficient.

Mortality and assessment of colonization. Inoculation (vaccination) with approximately 10⁷ CFU of live P. mirabilis HI4320 delivered via the urethra into the bladder resulted in the death of 16 (27%) of 60 mice. Of 30 sham-vaccinated (control) mice, none died over the next 35 days. Following 4 days of ampicillin administration, urine cultures of 4 of 44 surviving vaccinated mice were still positive for P. mirabilis HI4320, these 4 were not further evaluated. No bacteria were detected in day 35 urine cultures of sham-vaccinated mice. Of the 40 surviving animals whose P. mirabilis infection was eradicated by ampicillin, 10 were randomly assigned to a sham challenge group and received only PBS transurethrally. The rest of the vaccinated animals (n = 30) and the 30 sham-vaccinated animals were challenged with P. mirabilis Nal^r HI4320. Eight (27%) of the vaccination group and nine (30%) of the sham-vaccinated group died within 7 days after the challenge.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Colonization of the urine, bladder, and kidneys 7 days after transurethral challenge with P. mirabilis Nalr HI4320 in vaccinated () or sham-vaccinated () mice. The P values are for comparisons of mean numbers of CFU per milliliter or gram of urine or bladder or kidney homogenates from vaccinated and sham-vaccinated mice. The fractions above the bars are the numbers of mice colonized at a level of 103 CFU/ml or g of urine, bladder, or kidney over the total number of specimens examined.

Histopathology. On day 42, half of each kidney was processed for light microscopy and evaluated by a previously described procedure (10). Acute pyelitis and pyelonephritis were observed in 13 of 20 sham-vaccinated mice, with a mean histologic score of 1.48 ± 1.38 (standard error of the mean [SEM]). No mononuclear infiltrates or fibrosis was observed in the kidneys of those mice. Moderate-to-severe dilatation of the renal pelvis was also observed in those mice. In sham-challenged mice, no acute renal inflammation was observed. Mononuclear inflammation, predominantly lymphocytes (>90% of the cells) confined to the subepithelial connective tissue of the pelvis, was observed in 10 of 10 of those mice, with a mean histologic score of 1.8 ± 1.03 (SEM). There was mild (1+) fibrosis and mild (1+)-to-moderate (2+) dilatation of the pelvis. The renal parenchyma did not show fibrosis or scarring.

Antibody response to P. mirabilis. On days 0 and 35, mice were bled retro-orbitally and serum was evaluated by enzyme-linked immunosorbent assay (ELISA) for antibodies to whole P. mirabilis cells. Flat-bottom microtiter wells were coated with antigen prepared as a formalin-inactivated broth culture containing approximately 10⁸ CFU/ml of 0.06 M carbonate buffer, pH 9.6. After the wells had been washed three times with PBS containing 0.05% Tween 20 (PBS-T), the nonbinding sites in the wells were blocked by using 3% bovine serum albumin (BSA) in PBS. After the wells had been washed three times with PBS-T, serial twofold dilutions of serum in PBS-T containing 1% BSA were added to the wells and they were incubated at 37°C for 1 h. After the wells had been washed five times with PBS-T, alkaline phosphate-conjugated goat anti-mouse immunoglobulin (Ig) in PBS-T containing 1% BSA was added to the wells and they were incubated for 1 h at 37°C. After the wells had been washed five times with PBS-T, p-nitrophenylphosphate in 10% diethanolamine buffer, pH 9.8, was added to the wells and they were incubated for 30 min at 37°C. After the reaction had been stopped by addition of 3 N NaOH, the A₄₀₅ was read (9).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Serum anti-P. mirabilis responses in individual mice 35 days after transurethral vaccination with live P. mirabilis HI4320.

Antibody response to specific antigens. Isogenic mutants of P. mirabilis HI4320 were used for detection of serum antibodies directed against specific proteins, including MR/P fimbriae (2), PMF fimbriae (13), urease (11), and flagella (16), by Western blot analysis. For preparation of bacterial proteins, P. mirabilis strains were passaged three times in nutrient broth statically for 48 h at 37°C, conditions that favor the expression of MR/P fimbriae (1). The same cultures were used for preparation of PMF fimbriae. For preparation of flagella, bacteria were grown overnight on TSA plates. For preparation of urease, cultures were induced overnight with 100 mM urea. Whole-cell preparations from the wild-type strain and isogenic mutants were solubilized in sodium dodecyl sulfate (SDS)-gel sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore) as described by Towbin et al. (28). Immunoblots were developed with serum from vaccinated mice with elevated Ig titers. For preparation of MR/P fimbrial antigens, the whole-cell preparation was pretreated with 10% trichloroacetic acid, necessary to denature MR/P fimbriae (1), prior to solubilization in SDS-gel sample buffer.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Western blots using serum from a mouse 35 days after vaccination by transurethral challenge with P. mirabilis HI4320 and isogenic mutants. Shear preparations from P. mirabilis HI4320 (wild type) and its PMF-negative (A) or flagellum-negative (B) isogenic mutants were used to prepare Western blots. Sera from vaccinated mice were used as primary antiserum for detection of antigen.

A significant serum Ig response to P. mirabilis does not correlate with protection from homologous challenge. The live vaccination resulted in the development of measurable levels of IgG and IgM in the sera of 15 of 20 animals; only 4 animals developed detectable serum IgA levels. Others have reported increases in antibody responses following P. mirabilis vaccination. Pazin and Braude (21) reported the development of immobilizing serum antibody to H antigen following intravenous vaccination of rats with formalin-killed P. mirabilis. The immobilizing antibody prevented the spread of P. mirabilis infection of one kidney through the urinary tract to the uninfected kidney. Domingue et al. (4) reported substantial increases in hemagglutinin titers (Ig classes not defined) following intravenous vaccination of rabbits with enterobacterial common antigen which resulted in protection from both retrograde and hematogenous pyelonephritis. In our mice, although most of the animals developed measurable serum IgG and/or IgM titers following live vaccination, quantitative titers did not correlate with protection from infection or mortality. Indeed, for those animals with serum IgG levels of >1,000, five of six sacrificed on day 42 were infected; two of eight mice that died between days 35 and 42 had IgG titers of >1:1,000. On the whole, IgM titers were lower than IgG titers; however, the one animal with a very high IgM titer (1:20,480) became infected and five of eight mice that died had titers of 1:80. None of three mice with elevated IgA titers sacrificed on day 42 were infected, but one mouse that died on day 41 had a titer of 1:40.

Use of isogenic mutants to identify Ig responses to specific antigens. As demonstrated in this study, the use of isogenic mutants allows clear identification of the host immune response to specific antigens of the infecting organism through the use of cell lysates containing or not containing a target antigen. We are able to detect the time after challenge when specific bacterial surface antigens are recognized by the host immune response. These results provide information that will likely lead to a more effective immunization strategy by identifying bacterial antigens the host recognizes as important as evidenced by a postinfection antibody response. Vaccination with a purified targeted protein identified by this technique and perhaps the use of a more effective route of antigen presentation for antibody production may lead to enhanced protection from UTI. Proteus fimbriae, which might induce antibodies that block bacterial attachment to the uroepithelium, or flagella, which might induce antibodies that immobilize the organisms, may represent vaccine candidates.