Cyclophosphamide. was withdrawn by a needle and syringe, and the. samples were plated in molten agar. At the time of

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1 INFECTION AND IMMUNITY, Nov. 1982, P /82/ $2./ Copyright 1982, American Society for Microbiology Vol. 38, No. 2 Increased Severity of Urinary Tract Infection and Bacteremia in Mice with Urinary Bladder Injury Induced by Cyclophosphamide DAVID LYON,1 EDWIN B. HOWARD,2'3 AND JOHN Z. MONTGOMERIE4* Department of Medicine, Rancho Los Amigos Hospital, Downey, California ; Departments of Pathology2 and Medicine,4 University of Southern California School of Medicine, Los Angeles, California 933; and Laboratory and Disease Investigation Division, Comparative Medical and Veterinary Services, Los Angeles County Department of Health Services, Downey, California Received 3 May 1982/Accepted 14 July 1982 The effect of cyclophosphamide on urinary tract infection was studied, using Pseudomonas aeruginosa in a murine model. Urinary tract infections were produced by injecting P. aeruginosa through a urethral catheter into the bladders of mice. The number of P. aeruginosa organisms in the bladder tissue and kidneys, histopathology, peripheral leukocyte count, and antibody response to P. aeruginosa was measured. The local effect of cyclophosphamide on the bladder was determined by measuring the bladder tissue water and examining the histopathology. Cyclophosphamide increased the susceptibility of mice to P. aeruginosa urinary tract infection, resulting in marked cystitis and an increase in renal infection, bacteremia, and mortality. These changes correlated with the toxic effect of cyclophosphamide on the wall of the bladder rather than with peripheral leukopenia or failure of antibody response. Cyclophosphamide is an immunosuppressive drug used to treat leukemia, solid tumors, and other diseases, but serious infections may occur as a consequence of immunosuppression. Pseudomonas aeruginosa has been shown to be a significant pathogen in these infections. Cyclophosphamide has a number of potential effects on the immune system resulting in infection, of which leukopenia has been considered to be the most important (6). In the urinary tract, cyclophosphamide may also affect the wall of the bladder, resulting in hemorrhagic cystitis (1, 5, 7, 9). We examined the effect of cyclophosphamide on urinary tract infection with P. aeruginosa in mice and correlated the course of bacterial infection with histological changes in the bladder wall, peripheral leukocytes, and antibody responses. MATERIALS AND METHODS Production of P. aeruginosa urinary tract infections. The bacteria, mice, and methods of producing urinary tract infection were the same as those described previously (8). Swiss Webster female mice weighing 18 to 2 g each were infected by the injection of.25 ml of washed culture containing 5 x 16 cells of P. aeruginosa T2 into the bladder through a urethral catheter. At the time of sacrifice, blood was taken from the heart for serology and culture, and the number of bacteria in the kidneys and bladder were determined quantitatively. The urine in the bladder was withdrawn by a needle and syringe, and the kidneys and bladder were removed aseptically and homogenized in broth. Serial dilutions of the homogenates were made in brain heart infusion broth, and samples were plated in molten agar. At the time of sacrifice, sections of the bladder and kidneys were taken for histopathological examination. The histological examination was carried out blindly. The degree of edema, hemorrhage, polymorphonuclear leukocyte (PMN) and lymphocyte infiltration, ulceration, and bacteria in each section were each assessed semiquantitatively from to 3+, and the mean histological changes for each abnormality were compared by Student t statistics. Groups of five mice were to be examined at 4 h, 3 days, and 1, 2, and 4 weeks. Controls included normal mice infected with P. aeruginosa, mice treated with cyclophosphamide that received phosphate-buffered saline by urethral catheter, and normal mice that received phosphate-buffered saline by urethral catheter. Immune suppression with cyclophosphamide. Swiss Webster female mice were treated with cyclophosphamide (Cytoxan; Mead Johnson Laboratories). A dose of 1 mg of cyclophosphamide in saline per kg of body weight was given on alternate days for three doses. Control mice received saline only. All mice were injected by the intraperitoneal route. In a second experiment, one group of mice received 25 mg/kg as a single dose and were challenged with P. aeruginosa 5 days later, and another group of mice received 25 mg/kg daily for 1 days and were challenged with P. aeruginosa after three doses. Antibody determination. Serum and urine antibody titers were measured by indirect enzyme-linked im- 558

2 VOL. 38, 1982 munosorbent assay. The technique was adopted from the method of Holmgren and Svennerholm (4). Two antigens were used, a crude extract of P. aeruginosa T2 obtained by boiling a suspension of the bacteria and purified lipopolysaccharide. A semimicrosystem in which disposable polystyrene microcuvettes are used was employed. Alkaline phosphatase was conjugated to the immunoglobulin G fraction of rabbit anti-mouse immunoglobulin G, immunoglobulin M, and immunoglobulin A with gluteraldehyde. Urine collection. Groups of five mice were placed in metabolic cages, and their urine was collected under mineral oil for 24 h in containers with.1% sodium azide. The mineral oil and debris were separated by centrifugation, and the urine was stored at -7 C. Determination of peripheral leukocyte count. Blood was obtained by retro-orbital puncture to determine the leukocyte and differential counts. Total peripheral leukocytes were determined with a Coulter counter, and differential leukocyte counts were carried out on blood smears. Measurement of bladder water content. Mice were killed after the administration of cyclophosphamide and weighed. Any urine in the bladder was withdrawn by needle and syringe, and the bladder was removed. The bladders were weighed wet and after drying 18 h at 1 C. The water content was calculated as milligrams per 1 mg of body weight. CYCLOPHOSPHAMIDE, URINARY TRACT INFECTION RESULTS Preliminary study. In the first experiment, the mice were injected with 1 mg of cyclophosphamide per kg on alternate days for three doses, and P. aeruginosa was inoculated into the bladder through a urethral catheter 1 day after the last dose of cyclophosphamide. This experiment terminated prematurely because all of the infected animals treated with cyclophosphamide died within 3 days of infection. The mean total peripheral leukocyte count in the cyclophosphamide-treated mice at the time of challenge was 2.8 x 13, with <1 PMNs. Three days after infection, the mean leukocyte count had increased to 6.4 x 13. In cyclophosphamide-treated mice that did not receive an inoculation with P. aeruginosa, the mean leukocyte count had increased to 3.4 x 13 at 3 days. There was a marked increase in the number of P. aeruginosa isolates from the bladder and kidneys at 3 days in the cyclophosphamidetreated group, although there were no differences between the treated and control groups when the mice were first examined at 4 h. Infection of the bladders and kidneys of mice after injection of P. aeruginosa into the bladder through a urethral catheter confirmed previous histopathological studies showing inflammation of the bladder at 4 h and renal infection in approximately half of the kidneys within 3 days. The infection was self-limited. In mice treated with cyclophosphamide, there were marked histopathological changes first notx 8- Q7- I i-6 I < 5 > ~l. a 4hrs POST- INFECTION DAYS 4hrs POST-INFECTION DAYS 559 FIG. 1. Number of P. aeruginosa isolates in bladders (A) and kidneys (B) of cyclophosphamide-treated mice infected with P. aeruginosa. The doses of cyclophosphamide given were 25 (-), 25 (A), and () mg/kg. ed at 4 h that were most striking at 3 days. At that time, mice pretreated with cyclophosphamide had developed extensive lesions in the bladder and kidneys. These lesions consisted of a marked infiltration of the submucosa by PMNs, and there was a significantly greater amount of submucosal hemorrhage and ulceration of the mucosa with a dense infiltration of PMNs into the ulcerated areas. Mice receiving cyclophosphamide alone showed some vacuolization of epithelial cells and edema of the wall, but the bladders otherwise appeared normal by histological examination. A 14 B

3 56 LYON, HOWARD, AND MONTGOMERIE -I 2.] u M.5 z w hrs. 3 POST- INFECTON EDEMA PMN HEMORRHAGE BACTERIA DAYs ULCERATION [I] LYMPHOCYTES FIG. 2. Histopathological changes in the bladders of cyclophosphamide (CYCLO)-treated mice infected with P. aeruginosa. Asterisks indicate results that were significantly different from those of both other groups (P <.5). Experiment with high and low doses of cyclophosphamide. In another experiment, two doses of cyclophosphamide were used, and there was a significant change in the numbers of bacteria in the bladders and kidneys (Fig. 1A and B) of mice that received 25 mg/kg. At 3 days, increased numbers of bacteria were found in the bladders and kidneys of mice receiving 25 mg/kg, but at 1 week, the number of bacteria in the bladders and kidneys were not significantly different from those in infected control mice. Histopathological changes were significantly more marked only in mice receiving the higher dose of cyclophosphamide (Fig. 2). Increased edema, hemorrhage, PMNs, ulcers, and bacteria were seen in the bladder walls at 24 h and 3 days. The striking histological change was a marked PMN infiltrate in the bladder. Histological changes in the kidneys were not so striking. In mice receiving 25 mg of cyclophosphamide, there was evidence of kidney infection with PMNs in the kidney cortices and significantly greater numbers of PMNs in the kidney pelvises. No significant differences were seen at 7 or 14 days, and the renal infection was self-limited. Infected mice receiving 25 mg of cyclophospha- CYCLO 25 mide per kg did not behave differently from infected control mice. Control mice treated with 25 mg of cyclophosphamide per kg and receiving phosphate-buffered saline by urethral cathe- R ter showed edema of the bladder wall and some vacuolization of the bladder epithelial cells within the first few days. No abnormalities were seen in mice that received 25 mg of cyclophosphamide alone per kg. The PMN levels in the peripheral blood are CYCLO 25 x 1 shown in Fig. 3. The most marked neutropenia was seen in mice receiving 25 mg of cyclophosphamide per kg, and the number of PMNs remained low for 14 days. Mice receiving 25 mg of cyclophosphamide per kg were neutropenic only on the day of challenge with P. aeruginosa. After this time, there was a marked rebound in PMNs in infected and non-infected mice. NO CYCLO At 3 days, bacteremia was found in three of five mice that had received 25 mg of cyclophosphamide per kg but was not seen in mice receiving 25 mg/kg. The serum antibody levels in mice 7 14 infected with P. aeruginosa were not significantly different from uninfected control mice at all time intervals. At 14 days, however, there was an increase in serum antibody in infected mice 6-,; Z 2-2 I INFECT. IMMUN. CYCLO 25 x 1 NO CYCLO POST- INFECTION DAYS FIG. 3. Blood PMN counts in mice infected (closed symbols) and not infected (open symbols) with P. aeruginosa and treated with cyclophosphamide (CY- CLO). 14

4 VOL. 38, 1982 that had received 25 mg of cyclophosphamide per kg. Antibody to P. aeruginosa was not detected in the urine. Effect of cyclophosphamide on the bladder. To examine the effect of cyclophosphamide on the bladder, mice were given the three different doses of cyclophosphamide that were used in the two experiments before challenge with P. aeruginosa, i.e., 25 mg/kg in one dose, 25 mg/kg in three doses, and 1 mg/kg in three doses. The bladders were then examined at the same time at which the bladders were inoculated with P. aeruginosa in the previous experiments, i.e., 5 days after the single dose of 25 mg/kg, 1 day after the third dose of 25 mg/kg, and 1 day after the last dose of 1 mg/kg. Control mice received three.2-ml doses of saline at the same time at which the animals were given 25 and 25 mg/kg doses, and controls were examined 1 day after the last injection. The water content (expressed as milligrams per 1 mg of body weight) of the bladders of mice receiving 1 mg/kg in three doses (11.3 ± 25.7) and 25 mg/kg (18.3 ± 39.8) in a single dose was significantly greater (P <.5) than that of mice receiving 25 mg/kg (59. ± 12.9) or that of control mice (62.5 ± 7.8). DISCUSSION The effect of cyclophosphamide on P. aeruginosa urinary tract infection was examined, using a murine model. Ascending urinary bladder and renal infection with P. aeruginosa was produced by injecting bacteria into the bladder through a urethral catheter, without further manipulation of the urinary tract. As noted in previous studies (8), inflammation of the bladder developed rapidly, and self-limited renal infection occurred in more than half of the mice. Ulceration of the urinary bladder occurred in mice inoculated with P. aeruginosa and was associated with infiltration of the bladder wall with PMNs. Mice treated with high doses of cyclophosphamide developed severe lesions in the bladder, with marked edema, ulceration, and bacterial multiplication. Many mice receiving high doses also developed bacteremia; there was high mortality among these animals. Cyclophosphamide has two effects which may influence development of urinary tract infection acutely in mice. An effect of the metabolite acrolein, which is toxic to the bladder (2, 9), may influence the development of infection in the bladder. Also, cyclophosphamide causes leukopenia. We believe that cyclophosphamide mainly affected the bladder. Increased infection in the bladder and kidneys was associated with the high doses only. The exact nature of the bladder lesion produced by cyclophosphamide is not well understood, but edema seems to be the CYCLOPHOSPHAMIDE, URINARY TRACT INFECTION 561 most frequently described initial lesion in the bladder wall. Mice are relatively resistant to the effects of cyclophosphamide as compared with rats (7), and in the absence of infection, we observed only vacuolization of epithelial cells in some sections and transient edema at the high doses. Bladder wet weights, however, indicated that there was marked edema. In the first experiment, there was a marked leukopenia which may have played a role in the early death of the mice. In the second experiment, there was a brief leukopenia that quickly reverted with the infection and, indeed, the lesions in the bladder showed marked infiltration of the lesion with PMNs. In addition, leukopenia was more prolonged in mice receiving the low dose of cyclophosphamide than in mice receiving the high dose. The mice receiving the low dose did not have significantly more infection than control mice (number of bacteria per gram of tissue or histopathology). The rapid development of the lesion in 2 or 3 days and the failure to to detect significant amounts of serum antibodies makes it unlikely that antibody played a significant role in the disease process. The relevance of the findings of this study to clinical medicine is that the use of cyclophosphamide in patients with P. aeruginosa urinary tract infections may cause them to be predisposed to severe bladder infection, renal infection, and bacteremia. P. aeruginosa has been an important cause of hospital-associated infection. Special problems have been seen in immunosuppressed patients with leukemia or cancer. Although the urinary tract has been the most common site of P. aeruginosa infection in hospital environments, there have been few studies of the urinary tract as a potential portal of entry for infection in immunosuppressed patients. The increased incidence of urinary tract infection after renal transplantation has been associated with bacteremia (1). Cyclophosphamide is used not infrequently in these patients and should be examined as a possible factor is such infections. We have not found reports of severe urinary tract infection in patients receiving cyclophosphamide for leukemia or solid tumors. One report of 4 patients with bladder fibrosis and telangiectasia associated with cyclophosphamide therapy did not mention infection (5). Goldman and Warner did note cytomegalic inclusions in the bladder of patients with cyclophosphamide therapy (3). It is possible that severe bladder infection associated with bacteremia has not been documented in the past because of the difficulties in documentation. The bladder is not often biopsied, and changes at postmortem could be ascribed to cyclophosphamide cystitis. In addition, bacteremias are often considered to result

5 562 LYON, HOWARD, AND MONTGOMERIE from urinary tract infections, the exact sources being undocumented. ACKNOWLEDGMENTS We thank Jean Lloyd and Maureen Sanchez for their secretarial assistance and Donald G. Schick and Mary A. Ashley for technical assistance. LITERATURE CITED 1. Coggins, P. R., R. G. Ravdin, and S. H. Eisman Clinical pharmacology and preliminary evaluation of cytoxan (cyclophosphamide). Cancer Chemother. Res. 3: Cox, P. F Cyclophosphamide cystitis-identification of acrolein as the causative agent. Biochem. Pharmacol. 28: Goldman, R. L., and N. E. Warner Hemorrhagic cystitis and cytomegalic inclusions in the bladder associated with cyclophosphamide therapy. Cancer. 25: Holmgren, J., and A. M. Svennerholm Enzymelinked immunosorbent assays for cholera serology. Infect. Immun. 7: INFECT. IMMUN. 5. Johnson, W. W., and D. C. Meadows Urinarybladder fibrosis and telangiectasia associated with longterm cyclophosphamide therapy. N. Engl. J. Med. 284: Levine, A. S., S. C. Schimpf, R. G. Graw, and R. C. Young, Jr Hematologic malignancies and other marrow failure states: progress in the management of complicating infections. Semin. Hematol. 11: Levy, L., and R. Harris Effect of N-acetylcysteine on some aspects of cyclophosphamide-induced toxicity and immunosuppression. Biochem. Pharmacol. 26: Montgomerie, J. Z., W. J. Tuddenham, E. B. Howard, and J. W. Morrow Pseudomonas urinary tract infection in mice. Infect. Immun. 29: Philips, F. S., S. S. Sternberg, A. P. Cronin, and P. M. Vida Cyclophosphamide and urinary bladder toxicity. Cancer Res. 21: Rubin, R. H Infection in the renal transplant patient, p In R. H. Rubin, and L. S. Young (ed.), Clinical approach to infection in the compromised host. Plenum Medical Book Co., New York. Downloaded from on September 27, 218 by guest