Modulation of Candida albicans Attachment to Human Epithelial Cells by Bacteria and Carbohydrates

Transcription

1 INFECTION AND IMMUNITY, Mar. 1983, p /83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 39, No. 3 Modulation of Candida albicans Attachment to Human Epithelial Cells by Bacteria and Carbohydrates A. CENTENO,' C. P. DAVIS,2* M. S. COHEN,' AND M. M. WARREN' Department of Surgery, Division of Urology,' and Department of Microbiology,2 University of Texas Medical Branch, Galveston, Texas Received 22 July 1982/Accepted 21 December 1982 The effects of carbohydrates (mannose and dextrose), Escherichia coli 07KL, and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Urinary candidiasis is being detected with increasing frequency in nosocomial infections (9, 15, 32). Candida albicans is an opportunistic pathogen appearing in predisposed patients (e.g., diabetics, patients receiving chemotherapeutic or immunosuppressive medications) after bacterial infections have been treated (1, 15, 28, 31, 32). Past studies of bacteria-yeast interaction suggested that bacteria inhibit the growth of C. albicans in vitro (24) and decrease the ability of yeasts to infect the gut (11) and buccal (20) epithelium. Proposed mechanisms include: secretion by bacteria of antifungal substances (11, 24), competition for nutrients (11, 24), competition for cell receptors (20), and alterations of the environment by bacterial infections (11, 20, 24). Attachment of bacteria to epithelial cells is recognized as a crucial step of colonization and infection in the host organism (5, 8, 34, 36, 37). Studies of C. albicans attachment to oral and vaginal mucosal epithelial cells suggest that attachment is mediated by epithelial cell receptors containing mannose moieties similar to those involved in bacterial attachment (29). There have been studies showing bacterial pili agglutination of the fungus Saccharomyces cerevisiae, a close relative of C. albicans (6, 14, 18, 19). A thorough understanding of factors mediating yeast attachment, however, has not been attained. We are unaware of any studies investigating yeast attachment to human urothelial cells. The following study was conducted to investigate the effect of certain bacteria on yeast attachment to epithelial cells and to study those factors affecting bacterial and yeast attachment. This study provides new evidence for facilitation of C. albicans attachment to epithelial cells by certain piliated strains of urinary bacterial pathogens. (This work was submitted in partial fulfillment of the requirements for a Master of Science degree at the University of Texas Medical Branch, Galveston.) MATERIALS AND METHODS Growth and isolation of Candida organisms. C. albicans (a clinical isolate from John Sealy Hospital, Galveston, Tex., identified by the mycology laboratory) was grown on Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). The use of Sabouraud growth media yields predominately a yeast-phase organism (1, 16, 17, 35). After 48 h of incubation at 37 C, the yeasts were harvested, and the cells were suspended in 5 ml of phosphate-buffered saline (PBS, ph 7.2). These suspensions were washed three times with PBS (ph 7.2; 5 ml, 5 min, and 700 x g each time). The pellet was resuspended in 5 ml of PBS. The organisms were counted with a Petroff-Hausser counting chamber. The solution was diluted to a concentration of 10i yeast cells per ml of PBS and recounted. Epithelial cells. Human exfoliated urothelial cells were obtained by collecting freshly voided urine from human female volunteers. Buccal cells were collected by gently scraping the oral mucosa of a human male volunteer with sterile swabs and suspending the cells in 5 ml of PBS. These cell suspensions were centri- 1354

2 VOL. 39, 1983 C. ALBICANS ATTACHMENT TO EPITHELIAL CELLS 1355 fuged (700 x g, 15 min), washed three times with PBS, and resuspended to a concentration of 5 x 106 urothelial cells per ml of PBS, using a Petroff-Hausser counting chamber. While epithelial cells were being counted for final concentration, they were also examined for both preattached yeasts and bacteria. The presence of resident flora on epithelial cells was rare; attachment by yeasts was never observed, and attachment by bacteria was rarely observed. Cells with a resident flora were not used in the tests. Thus, washing the epithelial cells was quite effective in removing the vast majority of bacteria. Bacteria. The Klebsiella pneumoniae strain used throughout the study was obtained from the Clinical Laboratories of John Sealy Hospital, after isolation from a human urinary tract infection. The nonpiliated form of the organism was maintained on nutrient agar (BBL Microbiology Systems, Cockeysville, Md.) and stored at 4 C. The piliated phase was produced by serial passages in broth medium over the course of 1 week. Piliation was confirmed by hemagglutination of guinea pig erythrocytes (8). The piliated phase was maintained in Trypticase soy broth (BBL) with dextrose added at a concentration of 5 g/liter. The organisms were stored at -70 C until used. Serological tests with the organisms indicated that, antigenically, the phases differed only in the presence or absence of pili (7). No biochemical differences were noted in the two phases when the organisms were shifted from the nonpiliated to the piliated phase by serial passage in broth medium (7). To prepare bacteria for adherence studies, 24-h broth cultures in either piliated or nonpiliated phases were washed three times with PBS and resuspended in 5 ml of PBS to a concentration of 109 bacteria per ml of PBS. Escherichia coli 07KL has been described in detail previously (2). An inoculum of E. coli was incubated statically for 24 h in brain heart infusion broth (Difco). Organisms grown under these conditions routinely show pili with electron microscopy and show mannose-sensitive hemagglutination of guinea pig erythrocytes (3). Bacteria were harvested by centrifugation, washed three times with PBS, and resuspended in PBS to a final concentration of 109 bacteria per ml of PBS. Adherence studies. Epithelial cells (0.5 ml of a suspension of 5 x 106 cells per ml of PBS) and yeast organisms (0.5 ml of a suspension of 108 yeast cells per ml of PBS) were added to screw top vials (ratio of cells/yeasts, 1:20) and gently mixed with a Wrist Action Shaker (model 75; Burrell, Pittsburgh, Pa.) at 37 C for 60 min. After incubation, the suspensions were pipetted onto a 12-p.m pore size polycarbonate filter (Nuclepore Corp., Pleasanton, Calif.). Unattached yeasts were removed by rinsing the filters two times with 3 to 5 ml of pipetted PBS. This wash was adequate as background (free organisms on the filter unattached to epithelial cells) was minimal when assayed by light and scanning electron microscopy (SEM). The filters were labeled and either allowed to dry for 24 h at room temperature or placed directly into 2.5% glutaraldehyde with subsequent processing for electron microscopy. Light microscopy and staining. The dried filters were floated, epithelial cell side down, on the dye solution (methylene blue, 0.5% in PBS) for 10 to 15 s and then removed and replaced, epithelial cell side up, on the dye solution for 10 to 15 min. Excess dye was removed from the filters with three successive rinses by floating the filters in PBS-filled petri plates. After the final rinse, the filters were placed, epithelial side up, on large glass slides (3 by 2 in; Kimble, Toledo, Ohio) and viewed as wet mounts by light microscopy at x400. The first 30 epithelial cells viewed were counted for bacterial and yeast attachment. These counts were used for statistical analysis. Bacterial studies. Epithelial cells (0.5 ml of a suspension of 5 x 10' cells per ml of PBS) were mixed with either E. coli, piliated K. pneumoniae, or nonpiliated K. pneumoniae (0.5 ml of a suspension of 109 bacteria per ml of PBS) and incubated for 60 min at 37 C in a water bath with agitation utilizing a wrist action shaker. After incubation, the epithelial cells were pipetted onto filters, and the unattached bacteria were removed by washing as previously described. The filters containing epithelial cells with adherent bacteria were placed in petri plates with the side possessing epithelial cells facing up. Candida organisms (108 yeast cells per ml of PBS) were pipetted (0.5 ml) onto the surface of the filters. The filters were incubated in a humidity chamber for 60 min at 37 C. After incubation, the filters were washed as described above to remove unattached organisms. Carbohydrate studies. In the first series of experiments, either yeasts or bacteria were added to epithelial suspensions, constituting a control group (see Table 2). In the second series of experiments, epithelial cells were prepared in 0.5-ml solutions of dextrose (48 mg/ml of PBS) or mannose (12.5 mg/ml of PBS). Bacteria or yeasts were incubated with these epithelial cells in carbohydrate solution for 60 min. In the third series of experiments, bacteria were incubated with epithelial cells suspended in either a carbohydrate- or noncarbohydrate-containing solution. After 60 min, the epithelial cells were filtered free of unattached bacteria. Yeasts were then incubated for an additional 60 min with these epithelial cells in petri plates. Pilot studies indicated that the time difference (additional 60 min) between experiments showed essentially no alterations in attachment, so the three series could be compared. Carbohydrates were not subsequently added to yeasts and epithelial cells which had been preincubated with bacteria, as previous studies in our laboratory have demonstrated elution of attached bacteria (C. P. Davis, unpublished data). In all three series of carbohydrate-containing experiments, bacteria, yeasts, and epithelial cells were prepared as described above. SEM. Washed buccal epithelial cells were collected on polycarbonate filters. Consequently, only one major buccal cell surface was available for attachment. The cells were subsequently incubated with either bacteria, C. albicans, or both as described above. Filters to be examined by SEM were fixed for 24 h in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate. They were postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate. After 24 h, the filters were dehydrated with ethanol-water rinses (20, 40, 60, 75, 95, and 100% ethanol) at 15-min intervals. Critical point drying was performed in a critical point dryer (Polaron Instruments, Lexington, Ky.). Samples were then fixed to aluminum stubs, using silver paint, and coated with gold in an evaporator (Hummer II; Technics Inc., Alexandria, Va.). SEM was performed with

3 1356 CENTENO ET AL. INFECT. IMMUN. TABLE 1. Comparison of urothelial cells and buccal cells with or without carbohydrate incubation" Yeasts %b cell %d Cell type Yeasts %uid Yeasts per cell P value" ~~~~~Yeasts cell with per per cell %b P value' with mannose dextrose %si P value" Urothelial > P > P < NS Buccal Control P < NS a Yeast attachment to epithelial cells was viewed with light microscopy; mannose concentration was 12.5 mg/ ml; dextrose, 48 mg/ml. b Urothelial attachment percentage compared with buccal attachment of yeasts. ' Urothelial attachment versus buccal attachment of yeasts. d Urothelial percentage compared with urothelial and yeast incubation control experiment. Buccal percentage compared with buccal and yeast incubation control experiment. e Urothelial attachment compared with urothelial and yeast control experiment. Buccal attachment compared with buccal and yeast control experiment. NS, Not significant. an ISI Super III (ISI Inc., Mountain View, Calif.) at 15 kv Ṡtatistics. Statistical significance between means was determined by using a one-tailed Student's t test, based on experimental and control values found on the same day with cells or groups of cells from the same cell populations. RESULTS Epithelial cell adherence studies. Initial studies were performed to compare attachment of yeasts to urothelial and buccal cells. In general, adherence of C. albicans organisms were low (Table 1). The mean adherence ratio (yeasts to epithelial cell) (Table 1) compares favorably with those of Sandin et al. (29) and Kimura and Pearsall (16), who used buccal cells in their studies. Mean adherence was lower than that TABLE 2. found by Sobel et al. (35) and King et al. (17), who used predominately vaginal epithelial cells. Urothelial cells behaved in a qualitatively similar manner in attachment experiments; however, quantitatively, adherence ratios were 50% less than those calculated for buccal cells (Table 1). Control epithelial cells had no evidence of yeast contamination or attachment. Consequently, the levels of adherence noted on experimental cells could be measured accurately. Dextrose did not significantly alter yeast attachment to either group of epithelial cells (Table 1). Conversely, mannose significantly decreased yeast attachment to each group of epithelial cells (Table 1). Because of the difficulties encountered in obtaining exfoliated urothelial cells, the following experiments were done utilizing buccal cells. We found, as have other investigators (16, 17, Effect of bacteria and carbohydrate solutions on yeast attachment to epithelial cells Yeasts per % Incubation exptsa cell (mean + Attachment P value SEM) Yeast (control) 1.7 ± Control Yeast and mannose 0.5 ± b b Yeast and dextrose 1.5 ± b NSb Yeast and piliated K. pneumoniae 4.5 ± b <0.0005b Yeast, piliated K. pneumoniae, and mannose 1.1 ± ' <0.0005b Yeast, piliated K. pneumoniae, and dextrose 1.8 ± ' <0.OOOSC Yeast and nonpiliated K. pneumoniae 1.8 ± b NSb Yeast, nonpiliated K. pneumoniae, and mannose 0.6 ± <0.OOSd Yeast, nonpiliated K. pneumoniae, and dextrose 1.2 ± NSd Yeast (control) 2.6 ± Control Yeast and E. coli 9.2 ± " <0.0005b Yeast, E. coli, and mannose 3.2 ± e <0.oOOSe Yeast, E. coli, and dextrose 7.5 ± g NSe a K. pneumoniae and E. coli attachment studies were performed on different days and enumerated by direct counting. In those experiments utilizing carbohydrates, organisms were added after cell-carbohydrate suspensions had been prepared. b Compared with the yeast (control) group. NS, Not significant. ' Compared with the yeast and piliated K. pneumoniae group. d Compared with the yeast and nonpiliated K. pneumoniae group. e Compared with the yeast and E. coli group.

4 VOL. 39, 1983 C. ALBICANS ATTACHMENT TO EPITHELIAL CELLS 1357 FIG. 1. Yeast cell attachment after incubation with a group of buccal epithelial cells (control). Note cell borders marked with arrows. Bar, 5,um. 29, 35), a daily variance in yeast attachment. However, experiments repeated with the same protocol showed similar results when done on different days. In an attempt to discern if time of incubation increased adherence, incubation times were extended to 2 and 3 h to observe any changes in yeast attachment. No statistically significant difference was found in attachment ratios when incubation time was extended. Bacterial influence on Candida attachment. Yeasts attached in statistically greater numbers to epithelial cells that had been preincubated with piliated bacteriai strains (E. coli 07KL and heavily piliated K. pneumoniae) than to those epithelial cells that had not been preincubated with piliated bacterial strains (Table 2). The nonpiliated strain of K. pneumoniae did not alter attachment significantly. Mannose did significantly inhibit attachment of C. albicans to epithelial cells when added to incubation mixtures (Table 2). We also found that mannose inhibited piliated bacterial attachment to a significant degree but had no significant effect on nonpiliated bacterial attachment. Dextrose reduced attachment of yeasts when added to incubation solutions containing piliated K. pneumoniae (Table 2) but had no significant effect on nonpiliated K. pneumoniae or E. coli 07KL. SEM. SEM was performed to visualize the yeast-bacteria-epithelial cell interaction. The control group of epithelial cells showed single yeast attachment (Fig. 1). The epithelial cells preincubated with E. coli showed numerous bacteria attached to epithelial cells with bacteria juxtaposed to yeasts, singly and in clusters, on the epithelial cells (Fig. 2 and 3). In addition, similar bacteria-yeast interactions could be observed with organisms trapped on the filter surface and not attached to epithelial cells (Fig. 2). Piliated K. pneumoniae had the same microscopic appearance as E. coli with attached Candida organisms (Fig. 3); conversely, nonpiliated K. pneumoniae attached to epithelial cells, but yeasts were not found juxtaposed to bacteria (Fig. 4). In addition, fewer yeasts and bacteria were found on these cells when compared with cells preincubated with piliated bacteria. DISCUSSION The attachment of microorganisms is widely recognized as an initial step in establishing infection (2, 7, 17, 21, 23, 33). Many studies have shown that bacterial attachment to host cells can be augmented by pili (8, 27, 34, 36-38). Transmission electron microscopic studies of yeasts reveal that pili are not involved in fungal attachment when fungi alone are present in the test system (4, 12, 25, 26). Investigators have found that the outermost cell wall of C. albicans contains a floccular material with mannose-staining characteristics which are probably involved in yeast attachment (4, 13, 21). Sandin et al. (29) have found that extraction of mannans from the cell walls of Candida organisms prevented the yeasts from attaching to buccal cells. Evidence exists that suggests mannose moieties are present in epithelial cell surface receptors and are

5 1358 CENTENO ET AL. INFECT. IMMUN. FIG. 2. Piliated E. coli and associated yeasts attached to a group of buccal epithelial cells. In the lower right portion of the micrograph, note the adherence of yeast and bacteria together even when they are not attached to the epithelial cells (arrow). Bar, 5,um. involved to some extent with bacterial and yeast attachment (22, 29, 30). Previous investigations demonstrated that type 1 pili found on enterobacterial surfaces can exhibit mannose-sensitive agglutination of guinea pig erythrocytes and S. cerevisiae, a close relative of C. albicans (6, 10, 14, 18, 19). Our data show that either yeast or bacterial attachment alone is inhibited by mannose. This suggests that both may attach to a similar site on the epithelial cell. Our data also show an enhancement of yeast attachment by bacteria. This enhancement may be explained by a mechanism mediated by mannose-sensitive pili on the bacteria. This would explain the,4 o4 FIG. 3. Buccal epithelial cells with piliated K. pneumoniae and yeasts. Note the juxtaposition of yeasts and bacteria. Arrow denotes cell border. Bar, 2.5,um.

6 VOL. 39, C. ALBICANS ATTACHMENT TO EPITHELIAL CELLS 1359 FIG. 4. Buccal epithelial cells with nonpiliated K. pneumoniae. Note that yeasts and bacteria are not juxtaposed and that only one yeast is attached to the cells. Bar, 5 p.m. significantly increased attachment of yeasts to epithelial cells when piliated bacteria had been preincubated with the epithelial cells. The quantitative data is substantiated by SEM micrographs showing yeasts juxtaposed to piliated bacteria. Mannose inhibition of both piliated bacteria and yeasts is probably based on a competitive inhibition among mannose added to the incubation media, mannose-sensitive bacterial pili (type 1), and yeast surface mannans for receptor sites on the cell surface. In addition, it appears that some cell receptors and pili involved with piliated K. pneumoniae attachment may be competitively bound by dextrose, thus explaining the inhibition by dextrose of piliated K. pneumoniae bacterial and subsequent yeast attachment to epithelial cells. Dextrose is not as effective as mannose in inhibition, and this suggests that either cross-blocking with other carbohydrate receptor moieties occurs or that dextrose-sensitive receptors interact with pili and yeasts in the attachment process. These dextrose-sensitive receptors may be present but less important quantitatively than mannose-sensitive receptors. We hypothesize that the increase in yeast attachment to epithelial cells preincubated with piliated bacteria is due to an attachment of yeasts to mannose-sensitive pili on bacteria already attached to epithelial cells. This attachment may be an important factor in C. albicans pathogenicity. Piliated bacteria may facilitate Candida attachment, allowing the fungal organism a chance to form pseudohyphae and penetrate the host cell, in which it has been shown to grow and proliferate (12, 13, 21). Previously, colonization with bacteria was thought to inhibit fungal infections (11, 20, 24). Although this concept may be accurate in situations where the bacterial flora do not adhere to yeasts, our data suggests that certain piliated bacterial strains do significantly enhance yeast attachment to host epithelial cells. Currently, these in vitro observations are being extended to a rat model to test our hypothesis in vivo. Previous studies show that C. albicans, Torulopsis glabrata, and Candida tropicalis (both close relatives of C. albicans) are frequently found concomitantly with bacterial urinary tract infections (1). This adds clinical relevance to our study. Ultimately, better knowledge of yeastbacterial interactions may become important in the management of bacterial and candidal infections. ACKNOWLEDGMENTS This grant was supported in part by Public Health Service grants BSRG-S07-RR05427 and BSRG-S07-RR07205 from the National Institutes of Health. LITERATURE CITED 1. Ahearn, D. G., J. R. Jannach, and F. J. Roth Speciation and densities of yeasts in human urine specimens. Sabouraudia 5: Avots-Avotins, A. E., R. C. Fader, and C. P. Davis Environmental alterations and two distinct mechanisms of

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