Adherence of Gram-Negative Uropathogens to Human Uroepithelial Cells

0022-5347 /83/1302-0293$02.00/0 Vol. 130, August

THE JOURNAL OF UROLOGY

Printed in U.S.A.

Copyright© 1983 by The Williams & Wilkins Co.

ADHERENCE OF GRAM-NEGATIVE UROPATHOGENS TO HUMAN UROEPITHELIAL CELLS ANDREW W. BRUCE,* RAPHAEL C. Y. CHAN, DAVID PINKERTON, ALVARO MORALES AND PAUL CHADWICK From the Department of Urology, McGill University, Montreal, Quebec, Department of Biology, University of Calgary, Calgary, Alberta and Departments of Urology and Microbiology, Queen's University, Kingston, Ontario, Canada

ABSTRACT

The adherence of several gram-negative uropathogens to human uroepithelial cells was examined with scanning and transmission electron microscopy, and in vitro adhesion assays. The bacteria studied demonstrated different extracellular structures: fimbria and glycocalyx. Human uroepithelial cells were obtained from a bladder tumor cell line (T-24) and from different groups of patients, including those with no history of urinary tract infection (controls), recurrent urinary tract infection, acute cystitis and bladder tumors before and after bacillus Calmette-Guerin therapy. Results from these comparative studies showed that bacteria with extracellular structures adhered better than those bacteria without extracellular structures. It also was shown that uroepithelial cells obtained from patients with recurrent urinary tract infection and with acute cystitis were more susceptible to bacterial adherence than the uroepithelial cells of the controls (there were significant increases of 2 to 5-fold in mean adherence in the former 2 groups, p equals 0.015 and 0.002). There was no significant difference in bacterial adherence between the T-24 cells and the bladder tumor cells before bacillus Calmette-Guerin treatment. However, in both groups after treatment with bacillus Calmette-Guerin the mean adherence increased 2 to 4-fold (p equals 3 times 10-5 ). A survey of 29 bladder tumor patients also showed a 5-fold increase in the incidence of acquired urinary tract infection after bacillus Calmette-Guerin therapy. These results reveal a correlation between bacterial adherence and urinary tract infection, and suggest that bacterial adherence to uroepithelial cells depends upon the bacterial extracellular structures and the source of the uroepithelial cells. Adherence of bacteria to epithelial cells has been shown to have an important role in the pathogenesis of various infections in animals and in humans. 1 It is becoming increasingly apparent that bacterial adhesion to urinary tract epithelium is essential for initiation of a successful urinary tract infection2 ' 3 and researchers also have found a direct correlation between the increasing virulence of urinary tract infection and the increasing adhesiveness of the uropathogens to uroepithelial cells. 2 • 3 Various in vitro adhesion assay methods have been developed for studying the affinity of uropathogens for uroepithelial cells. 4 - 8 However, most of these studies have been performed on Escherichia coli, since this bacterium is responsible for a high percentage of nonspecific urinary tract infections in female patients. Various types of fimbriae 1• 4 and certain outer membrane components9 produced by the uropathogens have been reported to mediate adhesion, and specific receptors on the uroepithelial cell surface for fimbriae of E. coli have been identified. 10• 11 The adherence of the gram-negative uropathogens other than E. coli, especially the glycocalyx-producing isolates, to uroepithelial cells has received comparatively less attention, although it also has been reported that the bacterial glycocalyces are capable of mediating adherence. 12 Adhesion specificities of different E. coli strains to vaginal cells have been studied extensively4 • 6 ' 13 but the results remain contradictory. The variable abilities of uroepithelial cells from different groups of patients to accept bacteria also have been investigated4 • 5 • 14 and the results again are controversial. In this study we have used scanning and transmission electron microscopy, and in vitro adhesion assays to compare the adherence characteristics of several gram-negative uropathogens, demonstrating different extracellular structures to uroepithelial cells

obtained from different groups of patients. This study has allowed us to elucidate further the correlation between bacterial extracellular structures and adherence, and to clarify some of the controversial findings on the variable bacterial receptivities of uroepithelial cells obtained from different groups of patients. MATERIALS AND METHODS

Bacteria. All uropathogens selected for the study were isolated from urine specimens of patients with chronic cystitis, including 3 isolates of E. coli (1 fimbriated, 1 encapsulated and 1 without any electron microscopic evidence of an extracellular structure), 1 isolate of encapsulated Klebsiella pneumoniae and 2 isolates of Pseudomonas aeruginosa (rough and smooth colony types). The uropathogens were maintained on brain heart infusion agar slants and stored at -70C. The organisms were transferred to urine before use in adherence or electron microscopy studies. Growth and labeling of bacteria. All bacteria were grown at 37C unless otherwise stated. The uropathogens were prepared for isotopic labeling by overnight growth in filter-sterilized urine. The bacteria were then inoculated (1 per cent inoculum) into 2.0 ml. fresh, filter-sterilized urine supplemented with 20 uCi./ml. 3H-uridine, and incubated in an incubator-shaker at 100 revolutions per minute for 18 hours. The aforementioned bacterial cultures were harvested by centrifugation and the labeled bacteria were suspended to a concentration of 108 colony-forming units per ml. in phosphate buffered saline (pH 6.7). The ratio of colony-forming units to disintegrations per minute was determined by viable plate count and by collecting 0.2 ml. aliquots of labeled bacteria on a 0.2 um. pore size polycarbonate membrane filter. Uroepithelial cells and study groups. Uroepithelial cells Accepted for publication October 1, 1982. were obtained from freshly voided midstream urine of patients Supported in part by the Kidney Foundation of Canada. and controls in different study groups and from a bladder tumor * Requests for reprints: Toronto General Hospital, Room N6-249, Eaton Building North, Toronto, Ontario M5G 1L7, Canada. transitional cell line (T-24). The uroepithelial cells were har293

294

BRUCE AND ASSOCIATES

vested by centrifugation, washed once in phosphate buffered saline (pH 6. 7) and suspended in phosphate buffered saline to a concentration of 105 per ml. using a hemocytometer. The study groups included 4 healthy women (24 to 36 years old, mean 26.2), 4 women with a history and microbiological evidence of recurrent urinary tract infection (~2 episodes of infection per year) but who were not infected during the study (24 to 36 years old, mean 23.5), 4 women with acute cystitis (24 to 36 years old, mean 26.0), 4 patients (2 men and 2 women) with superficial bladder tumors before and after therapy with intravesical bacillus Calmette-Guerin (BCG). The T-24 cell line before and after addition, and incubation with BCG (40.0 mg./ ml., 37C, 2 hours) also was used. In vitro adhesion assay. The in vitro adhesion assay method was that of Schaeffer and associates, 6 with modifications. Briefly, aliquots of 0.2 ml. labeled bacteria and 0.2 ml. uroepithelial cells were combined and incubated at 37C (pH 6.7) for 30 minutes. Control tubes containing bacteria in phosphate buffered saline or uroepithelial cells in phosphate buffered saline also were prepared. After incubation 0.2 ml. of the mixture was filtered through a 5.0 um. pore size polycarbonate membrane filter. The filters then were washed with 20 ml. phosphate buffered saline (pH 6.7), air-dried, placed in scintillation vials containing 15 ml. scintillation fluid and counted in a minibeta counter. All experiments were run in duplicate. The number of bacteria adherent to uroepithelial cells retained on the duplicate filters was counted using a light microscope before scintillation counting. The number of adhering bacteria was determined by the counts per minute on the 5.0 um. filter of the mixture minus the counts per minute on the filter of the control. Adherence was calculated by dividing the number of bacteria adhering by the number of uroepithelial cells used in the assay. The adherence value was expressed as the number of bacteria adhering per uroepithelial cell. The mean adherence in the different study groups was analyzed by the paired t test. Transmission electron microscopy. Negative staining: Bacteria from the urine cultures were harvested by centrifugation, washed once in phosphate buffered saline (pH 7.2) and suspended in phosphate buffered saline. A drop of the bacterial suspension in phosphate buffered saline was applied to Formvar-coated copper grids (400 mesh), stained in 1 per cent phosphotungstic acid (pH 7.2) and allowed to air-dry before examination. Ruthenium red staining and embedding: The urine specimens and the in vitro adhesion assay mixtures were fixed in 5 per cent glutaraldehyde and cacodylate buffer (0.1 M, pH 7.2) with 0.15 per cent ruthenium red for 2 hours at room temperature. The preparations then were washed 5 times in the buffer, postfixed in 2 per cent osmium tetroxide in the buffer, washed 5 more times in the buffer and dehydrated through a graded acetone series. All of the solutions used in processing these specimens, from the washes following glutaraldehyde fixation to the 70 per cent acetone solution, were made up to contain 0.05 per cent ruthenium red. Ruthenium red was omitted from the 90 and 100 per cent acetone because of its limited solubility in these solutions. After further dehydration in propylene oxide the specimens were embedded in Vestopal, sectioned and stained with uranyl acetate and lead citrate. 15 All the prepared specimens were examined with a Hitachi 500 electron microscope at an accelerating voltage of 60 kv. Scanning electron microscopy. The infected urine and the in vitro adhesion assay mixtures were filtered through a 5.0 um. pore size membrane filter and washed with 20 ml. phosphate buffered saline (pH 6. 7). The specimens on the membrane filter were then fixed in 5 per cent glutaraldehyde and cacodylate buffer (0.01 M, pH 7.2) for 2 hours at room temperature. The fixed specimens were then washed 5 times in the cacodylate buffer, post-fixed in 1 per cent osmium tetroxide for 2 hours, washed 5 more times in distilled water and transferred to a 1 per cent thiocarbohydrazide solution for 30 minutes. The specimens were washed again 5 times in distilled water and the

post-fixation, and thiocarbohydrazide procedures were repeated twice before the specimens were dehydrated through graded ethanol-water and graded Freon-ethanol series. The dehydrated specimens were then critical point dried, mounted on stubs and sputter-coated with gold-palladium in a Hummer I sputter-coater for 1 minute. The specimens were then examined on a Hitachi 450 scanning electron microscope at an accelerating voltage of 20 kv. Survey of acquired urinary tract infection in patients with superficial bladder tumor before and after ECG therapy. To find the incidence of acquired urinary tract infection before and after BCG treatment a survey of 29 bladder cancer patients was undertaken, including 19 men and 10 women. The diagnosis of acquired urinary tract infection in these patients was made by the development of irritative urinary symptoms and the occurrence of positive bacterial urine cultures. RESULTS

Electron microscopic examination of the gram-negative uropathogens from infected urine specimens in vivo or from urine cultures in vitro demonstrated that these uropathogens can be categorized into 4 major morphological groups according to their abilities to produce different types of extracellular structures. The uropathogens may be fimbriated (fig. 1, A), encapsulated (fig. 1, B), slime-producing (fig. 1, C) or may demon-

Fm. 1. Transmission electron micrographs of urinary isolates. A, E. coli grown in urine, negatively stained with phosphotungstic acid. Note presence of fimbriae (arrowheads). Bar represents 0.5 um. B, thin section of ruthenium red stained cells of K. pneumoniae grown in urine. Note presence of integral fibrous glycocalyx (capsule) (S). Bar represents 0.25 um. C, thin section of ruthenium red stained cells of P. aeruginosa grown in urine. Note presence of flexible peripheral glycocalyx (slime) (S). Bar represents 0.25 um. D, thin section of ruthenium red stained cells of E. coli isolate grown in urine. Note absence of extracellular structures. Bar represents 0.25 um.

BACTERIAL ADHERENCE TO HUMAN UROEPITHELIAL CELLS

strate no extracellular structures on electron microscopy (fig. 1, D). When urine specimens obtained from patients with urinary tract infection were examined with transmission and scanning electron microscopy the bacterial extracellular structures, especially the bacterial glycocalyces in the form of capsule or slime, were found to mediate attachments between the uropathogens and the uroepithelial cells (fig. 2). It should be noted that the bacterial glycocalyx, in the form of capsule or slime observed by electron microscopy, usually appeared as a condensed, fibrous matrix (figs. 1, B and 2, A) or as collapsed fibrous material (fig. 1, C) surrounding the bacterial cells and interconnecting the bacterial cells to form adherent microcolonies (fig. 2, B). Examination by electron microscopic techniques of the in vitro adhesion assay mixtures of uropathogens and uroepithelial cells also revealed that adherence of the bacteria to uroepithelial cells was mediated by the bacterial extracellular structures (fig. 3). The fibrous bacterial glycocalyces again appeared as condensed masses on the bacterial cell surface (fig. 3, A) but were visualized occasionally as thin, extended fibers interconnecting bacteria to bacteria and bacteria to uroepithelial cells. Results from the in vitro adhesion assays revealed that adherence of uropathogens to uroepithelial cells increased by approximately 15 per cent when the pH of the assay mixture was at 4.5 and that no significant difference in adherence was noted when the assay temperature varied between lOC and

Fm. 3. A, transmission electron micrograph of thin section of ruthenium red stained bacteria and uroepithelial cell from in vitro adhesion assay mixture. Note adhesion of uropathogen (E. coli) (B) to uroepithelial cell (C) mediated by extracellular fibers (arrowhead). Bar represents 0.25 um. B, scanning electron micrograph of in vitro adhesion assay mixture of K. pneumoniae (B) and uroepithelial cells (C). Note that adhesion is mediated by fibrous material. Bar represents 2.0 um.

Fm. 2. A, transmission electron micrograph of thin section of ruthenium red stained urine specimen obtained from patient with E. coli infection shows glycocalyx-mediated adherence of uropathogen (B) to uroepithelial cell ( C). Note presence of fibrous capsule (S). Bar represents 0.5 um. B, scanning electron micrograph of infected urine from patient infected by encapsulated K. pneumoniae. Note presence of uropathogens in microcolonies (B) adhering to uroepithelial cell (C). Bar represents 2.0 um.

40C. Examination by light microscopy of the adherent bacteria on the membrane filters demonstrated that the fimbriated E. coli isolate attached only to the uroepithelial cells, while the glycocalyx-producing uropathogens attached to the uroepithelial cells and to the uromucoid. Quantitation of bacterial adherence by light microscopic and isotope assays correlated closely (± 5 per cent). Studies of bacterial adherence to uroepithelial cells versus time revealed that adherence generally was established within the first minute of contact between the bacteria and the uroepithelial cells, and then gradually declined to a stationary level (figs. 4 to 6). However, fluctuation or increase of adherence with respect to time also was observed, especially for those bacterial isolates that produced glycocalyces in the form of capsule or slime (figs. 4, C and 6, B). When the adherence characteristics of different morphological groups of uropathogens to uroepithelial cells were compared, using the in vitro adhesion assay, it was shown that the slimeproducing P. aeruginosa (the rough and the smooth colony types) had the highest degree of adherence, followed by the fimbriated E. coli and the encapsulated isolates of E. coli and K. pneumoniae while E. coli without any electron microscopicdemonstrable extracellular structures had the lowest adherence capability (figs. 4 to 6 and table 1). The adherence capability of uroepithelial cells obtained from different study groups was examined. Although there were slight day-to-day variations noted in the uroepithelial cells of the same study group in accepting uropathogens the difference

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FIG. 5. Graphical representations of in vitro adherence of uropathogens to uroepithelial cells obtained from bladder cancer patients before (A) and after (B) ECG therapy versus time. Results shown represent average of 4 duplicate experiments. 0----, E. coli, control. ~ E. coli, fimbriated. 0----, E. coli, encapsulated. 6 ...... , K. pneumoniae, encapsulated. D----, P. aeruginosa, rough colony type. II---, P. aeruginosa, smooth colony type. Latter 2 isolates produced slime when grown in urine.

in the mean adherence of uropathogens to uroepithelial cells obtained from the same study groups was insignificant. Uroepithelial cells obtained from sterile urine specimens of patients with a history of irritative bladder symptoms and bacteriological evidence of recurrent urinary tract infection were found to accept twice as many bacteria when compared to the uroepithelial cells obtained from healthy women (p = 0.015) (fig. 4 and table 1). The mean adherence of uropathogens to the uroepithelial cells of patients with acute cystitis was found to increase by 2 to 5-fold compared to the control group (p = 0.002). The mean adherence of the various uropathogens to uroepithelial cells obtained from patients with superficial bladder tumors before local instillation of BCG was found to be slightly higher than to the uroepithelial cells of the healthy controls (table 1) but the mean bacterial adherence to the uroepithelial cells of the bladder cancer patients after BCG therapy demonstrated a 2-fold elevation (p = 3 X 10- 5 ) (fig. 5

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FIG. 6. Graphical representations of in vitro adherence of uropathogens to uroepithelial cells obtained from T-24 cell line (A) and T-24 cell line after adding BCG (B) versus time. Results shown represent average of 4 duplicate experiments. 0----, E. coli, control. e------, E. coli, fimbriated. 0----, E. coli, encapsulated. 6 ...... , K. pneumoniae, encapsulated. D----, P. aeruginosa, rough colony type. II---, P. aeruginosa, smooth colony type. Latter 2 isolates produced slime when grown in urine.

and table 1). When the adherence of the uropathogens to a bladder tumor cell line (T-24) was studied and compared to the adherence pattern of the same uropathogens to the uroepithelial cells of the bladder cancer patients before BCG therapy it was noted that there was no significant bacterial receptivity difference between the 2 types of cells (p = 0.14) (table 1). However, there was again a 2 to 4-fold increase in bacterial adherence between the T-24 cells after the addition of the BCG vaccine (p = 0.005) (fig. 6 and table 1). To find the incidence of acquired urinary tract infection before and after BCG treatment a survey of 29 bladder cancer patients receiving BCG was undertaken (table 2). Of the 19 men surveyed 1 had had a previous urinary tract infection but after BCG therapy 8 became infected (42 per cent). Of the 10 women receiving BCG instillation 9 had urinary tract infection after treatment (90 per cent), while only 1 of these women had had a prior urinary tract infection. Thus, there was an over-all

297

BACTERIAL ADHERENCE TO HUMAN UROEPITHELIAL CELLS TABLE

1. In vitro adherence of uropathogens to different groups of uroepithelial cells No. Adherent Bacteria per Uroepithelial Cells* Control 5.3 ± 11.5 ± 11.5 ± 7.6 ± 22.4 ± 14.8 ±

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0.4 2.1 1.7 0.6 1.3 1.1

Recurrent Infection 11.9 ± 24.9 ± 25.1 ± 12.8 ± 44.0 ± 81.4 ±

Acute Cystitis

2.4 4.2 4.8 3.3 5.5 24.1

17.2 93.4 63.0 83.9 62.0 39.8

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6.2 40.0 24.8 32.9 13.6 12.4

Bladder Tumor Before BCG Therapy 6.5 20.4 14.9 9.3 24.1 37.7

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2.2 8.1 5.4 2.6 10.5 6.0

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3.5 9.7 6.5 6.0 12.0 7.8

T-24 Cell Before Adding BCG 5.4 12.7 18.7 10.5 32.0 42.5

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T-24 Cell After Adding BCG 20.6 41.3 42.1 28.9 52.9 116.0

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13.0 17.0 18.0 19.0 26.0 105.0

Control/recurrent infection p = 0.015, control/acute cystitis p = 0.002, bladder tumor before ECG/bladder tumor after BCG p = 3 x 10-5 , T-24 cell before BCG/T24 cell after BCG p = 0.005, bladder tumor before BCG/T-24 cell before BCG p = 0.14, bladder tumor after BCG/T-24 cell after BCG p = 0.05. * Mean adherence ± standard deviation. t Both isolates produced slime when grown in urine.

TABLE

2. Survey on bladder cancer patients acquiring urinary tract

infection before and after BCG therapy. Pts. Acquiring Urinary Tract Infection

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5-fold increase (10 to 58 per cent) in incidence of acquired urinary tract infection in those patients receiving BCG therapy. DISCUSSION

The female urinary tract is one of the most common sites for bacterial infection. Various gram-negative bacteria have been found to be the infecting pathogens, including E. coli, Proteus mirabilis, Proteus vulgaris, K. pneumoniae, P. aeruginosa and Serratia species. It has been demonstrated that most of the urinary isolates of E. coli are capable of producing various types of fimbriae to mediate their adherence to uroepithelial cells. 1' 4 Other gram-negative uropathogens that produce glycocalyces of polysaccharide nature have been reported to have an important role in adherence as well as in the pathogenesis of various infections, 12 although their importance in the pathogenesis of urinary tract infection has received less attention. By using scanning and transmission electron microscopy we have demonstrated that uropathogens that produce capsule or slime are capable of adhering to uroepithelial cells through the mediation of their glycocalyces. As noted the bacterial glycocalyces often were visualized as condensed or collapsed ruthenium red stained fibrous material because the exopolysaccharides are subjected to dehydration during preparation for electron microscopic examinations. 12 Results from our in vitro adhesion studies also have revealed that fimbriated and glycocalyx-producing uropathogens can adhere to uroepithelial cells. The mechanisms and specificities of bacterial adherence mediated by various types of fimbriae have been studied extensively1' s, 10' 11 and it is now clear that the mannose-sensitive fimbriae bind specifically to uromucoid, 8' 10 while the mannoseresistant fimbriae recognize a specific glycosphingolipid on the uroepithelial cell surface as the receptor. 11 However, the adherence mediated by the bacterial glycocalyces appears to be nonspecific 12 since the glycocalyx-producing uropathogens are found to attach to uroepithelial cells and to uromucoid. The actual mechanism of this type of adherence still requires further investigation. The adhesion specificity of various E. coli strains to vaginal and buccal cells obtained from different groups of patients has been examined4 • 5• 13· 14 but the variation in the ability of uroepithelial cells to accept uropathogens still remains controversial. Some investigators have found that uroepithelial cells from patients with recurrent urinary tract infection are more susceptible to bacterial adherence4· 14 and that susceptibility of patients to urinary tract infection is affected by their P blood

type, 16 whereas others have reported no difference in the adherence of E. coli 04 to the vaginal cells of normal and cystitisprone women. 5 Our results indicate that uroepithelial cells obtained from different study groups do demonstrate different susceptibilities to bacterial adherence. We have found that bacterial adherence to uroepithelial cells increases in patients with recurrent urinary tract infection and with acute cystitis when compared to women with no history or evidence of urinary tract infection, suggesting that the former 2 groups of patients are indeed cystitis-prone. In addition, patients with superficial bladder tumors receiving prophylactic BCG instillation were found to have a higher incidence of acquired urinary tract infection after BCG therapy. This increased incidence in acquired urinary tract infection correlated with the increased in vitro bacterial adherence to the uroepithelial cells obtained from these patients. In addition increased bacterial adherence was noted in the T-24 bladder tumor cell line after the addition of BCG. Although the increased incidence of infection in the BCG treated patients may be related to other factors, such as catheterization, 17' 18 the striking correlation between the increased incidence of urinary tract infection in patients following BCG treatment and the increased bacterial adherence to the uroepithelial cells of the same group of patients may provide further evidence that recurrent urinary tract infection is related to the bacterial receptivity of the uroepithelial cells. In conclusion, our results indicate that fimbriated and glycocalyx-producing gram-negative uropathogens adhere readily to uroepithelial cells and that bacterial adherence becomes more effective if the uroepithelial cells are from cystitis-prone patients (those with recurrent urinary tract infection), from patients with acute cystitis and from patients receiving intravesical BCG treatment. It would appear that bacterial adherence to uroepithelial cells depends upon the bacterial extracellular structures and the source of the uroepithelial cells. Mrs. V. Martinez and Dr. A. Pang provided technical assistance. REFERENCES 1. Beachey, E. H.: Bacterial adherence. London: Chapman & Hall, pp.

1 and 185, 1980. 2. Kiillenius, G. and Winberg, J.: Bacterial adherence to periurethral epithelial cells in girls prone to urinary-tract infections. Lancet, 2: 540, 1978. 3. Svanborg-Eden, C., Hanson, L. A., Jodal, U., Lindberg, U. and Akerlund, A. S.: Variable adherence to normal human urinarytract epithelial cells of Escherichia coli strains associated with various forms of urinary-tract infection. Lancet, 2: 490, 1976. 4. Svanborg Eden, C.: Attachment of Escherichia coli to human urinary tract epithelial cells. An in vitro test system applied in the study of urinary tract infections. Scand. J. Infect. Dis., suppl. 15, 1978. 5. Parsons, C. L., Anwar, H., Stauffer, C. and Schmidt, J. D.: In vitro adherence of radioactively labeled Escherichia coli in normal and cystitis-prone females. Infect. Immun., 26: 453, 1979. 6. Schaeffer, A. J., Amundsen, S. K. and Schmidt, L. N.: Adherence of Escherichia coli to human urinary tract epithelial cells. Infect. Immun., 24: 753, 1979.

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7. Mardh, P. A., Colleen, S. and Hovelius, B.: Attachment of bacteria to exfoliated cells from the urogenital tract. Invest. Urol., 16: 322, 1979. 8. Chick, S., Harber, M. J., MacKenzie, R. and Asscher, A. W.: Modified method for studying bacterial adhesion to isolated uroepithelial cells and uromucoid. Infect. Immun., 34: 256, 1981. 9. Ohman, L., Normann, B. and Stendahl, 0.: Physiochemical surface properties of Escherichia coli strains isolated from different types of urinary tract infections. Infect. Immun., 32: 951, 1981. 10. Orskov, I., Ferencz, A. and Orskov, F.: Tamm-Horsfall protein or uromucoid is the normal urinary slime that traps type 1 frmbriated Escherichia coli. Letter to the Editor. Lancet, 1: 887, 1980. 11. Leffler, H. and Svanborg-Eden, C.: Glycolipid receptors for uropathogenic Escherichia coli on human erythrocytes and uroepithelial cells. Infect. Immun., 34: 920, 1981. 12. Costerton, J. W. and Irvin, R. T.: The bacterial glycocalyx in nature and disease. Ann. Rev. Microbiol., 35: 299, 1981. 13. Fowler, J.E., Jr. and Stamey, T. A.: Studies ofintroital colonization in women with recurrent urinary tract infections. X. Adhesive properties of Escherichia coli and Proteus mirabilis: lack of correlation with urinary pathogenicity. J. Urol., 120: 315, 1978. 14. Schaeffer, A. J., Jones, J. M. and Dunn, J. K.: Association of in vitro Escherichia coli adherence to vaginal and buccal epithelial cells with susceptibility of women to recurrent urinary tract infections. New Engl. J. Med., 304: 1062, 1981. 15. Reynolds, E. S.: The use of lead citrate at high pH as an electron-

opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963. 16. Lomberg, H., Jodal, U., Svanborg-Eden, C., Leffler, H. and Samuelsson, B.: Pl blood group and urinary tract infection. Letter to the Editor. Lancet, 1: 551, 1981. 17. Stamm, W. E.: Infections related to medical devices. Ann. Intern. Med., suppl., 89: 764, 1978. 18. Garibaldi, R. A., Burke, J. P., Dickman, M. L. and Smith, C. B.: Factors predisposing to bacteriuria during indwelling urethral catheterization. New Engl. J. Med., 291: 215, 1974. EDITORIAL COMMENT This report provides further support for the concept that successful invasion of the urinary tract by pathogenic bacteria involves binding of the bacterium to the uroepithelium. This interaction appears to be mediated by the surface components on both cell types. The adhesive characteristics of bacterial strains and the receptivity of uroepithelial cells frequently correlate with the bacterial virulence in and host susceptibility to urinary tract infections. Nevertheless, the fact that some virulent strains are nonadherent and uroepithelial cells from patients with recurrent urinary tract infection frequently are nonreceptive suggests that additional factors have equally important roles in the pathogenesis of urinary tract infections. Anthony J. Schaeffer Department of Urology Northwestern University Medical School Chicago, Illinois