Adhesion, Hemagglutination, and Virulence of Escherichia coli Causing Urinary Tract Infections

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1 INFECTION AND IMMUNITy, Feb. 1981, p /81/ $02.00/0 Vol. 31, No. 2 Adhesion, Hemagglutination, and Virulence of Escherichia coli Causing Urinary Tract Infections LARS HAGBERG,1 ULF JODAL,"2 TIMO K. KORHONEN,3 GUNILLA LIDIN-JANSON,' ULF LINDBERG,2 AND CATHARINA SVANBORG EDAN'* Department of Pediatrics2 and Department of Clinical Immunology,' Institute ofmedical Microbiology, University of Goteborg, Goteborg, Sweden, and Department of General Microbiology, University of Helsinki, Helsinki, Finland' The capacity of 453 Escherichia coli to agglutinate erythrocytes and yeast cells and to attach to human urinary tract epithelial cells was tested. The were isolated from the urine of patients with acute pyelonephritis, acute cystitis, or asymptomatic bacteriuria and from the stools of healthy school children. Three main patterns of hemagglutination were found: (i) mannoseresistant agglutination of human erythrocytes alone or simultaneously with mannose-sensitive agglutination of guinea pig erythrocytes: (ii) only mannosesensitive agglutination of guinea pig and other erythrocytes; and (iii) no agglutination. Strains with mannose-resistant agglutination of human erythrocytes alone or in combination with mannose-sensitive hemagglutination attached in high numbers to human urinary tract epithelial cells. Bacteria inducing only mannosesensitive hemagglutination attached in low numbers, and non-agglutinating did not bind to the urinary tract epithelial cells. The bacterial surface antigen(s) mediating mannose-resistant hemagglutination of human erythrocytes and attachment to human urinary tract epithelial cells may be one factor selecting for E. coli from among the fecal flora which infect the urinary tract. The highest proportion of with this property was found among acute pyelonephritis isolates (77%), and the lowest proportion of with this property was found among normal fecal E. coli (16%). 564 Pili or fimbriae are nonflagellar filamentous bacterial surface appendages composed of hydrophobic proteins (1, 5, 12). Piliated bacteria stick to surfaces, both inorganic latex particles and organic animal or plant tissue (2, 3, 33). Besides bacterial binding due to this general "stickiness" (1), specific attachment to certain hosts and tissues occurs (3, 9, 28) and is thought to be a virulence factor for bacteria colonizing or causing infection of mucous surfaces (9). The role of pill as specific bacterial adhesins is presently under investigation. Bacterial adhesins have been classified according to the agglutination patterns resulting when bacteria bind to erythrocytes from various species. Bacteria causing agglutination of guinea pig erythrocytes inhibited in the presence of D- mannose are defined as carriers of type 1 pili (3, 5). Such pili are found on most enterobacteria without any obvious relation to the virulence of the (1, 3-5). The adhesins of another group of Escherichia coli cause agglutination of human erythrocytes which is unaffected by D- mannose. These mannose-resistant adhesins may or may not be of a pilus morphology (4) and have been associated with human diarrheal disease and urinary tract infection (UTI) (6, 7, 16, 30; C. Svanborg Eden, L. Hagberg, L. A. Hanson, T. K. Korhonen, H. Leffler, and S. Olling, Ciba Found. Symp., in press). For E. coli causing UTI in children the capacity to attach to human urinary tract epithelial cells in vitro was related to the severity of the UTI produced by the strain in vivo (31). The presence of pili on the, as seen by electron microscopy, and the capacity to attach to human urinary tract epithelial cells were strongly correlated (30). Although the pili were morphologically similar to type 1 pili, the attachment of the piliated was unaffected by D-mannose (30). Strains carrying only type 1 pili attached poorly to human urinary tract epithelial cells, whereas carrying mannose-resistant agglutinins attached in high numbers (16; T. K. Korhonen and C. Svanborg Eden, submitted for publication). Isolated pili and whole piliated bacteria showed identical hemagglutination and adhesion patterns (15, 27). The aim of the present study was to register the hemagglutination patterns of E. coli isolated from children with various forms of UTI or from the stools of healthy children and to search for a relation

2 VOL. 31, 1981 between virulence, hemagglutination pattern, and adhesion. MATERIALS AND METHODS Bacteria. A total of 453 E. coli were analyzed; 111 were isolated from the urine of 111 girls with their first known attack of acute pyelonephritis, 103 were isolated from 103 girls with acute cystitis, 119 were isolated from 119 girls with asymptomatic bacteriuria detected at screening, and 120 were isolated as the dominating fecal E. coli strain from the stools of non-bacteriuric school children (13, 19, 20). For diagnostic criteria see references 13 and 20. The were isolated between 1969 and 1974 and were kept in deep agar stab cultures until used. For culture, a liquid growth medium was selected so as not to prejudice the study in favor of a special adhesin. Bacteria were transferred from deep agar to Luria broth (22) (5 ml in a 100-ml flask), grown statically serially twice for 48 h, harvested by centrifugation, and suspended in phosphate-buffered saline (PBS) (300 mosm/liter, ph 7.1) to a concentration of about 2 x 10i bacteria per ml. The same bacterial suspension was used for agglutination and adhesion testing. Agglutination tests. Tests for agglutination were performed as described earlier (14). Suspensions (3%) in PBS of washed human, guinea pig, or horse erythrocytes or fresh bakers' yeast cells (Saccharomyces cerviciae) were mixed on a glass slide with PBS with or without D-mannose and with the bacterial suspension (20 pl of each). Agglutination was read immediately and after 10 min at 4 C, and graded -, +, or ++. The strength of the agglutination was determined further by serial twofold dilutions in PBS of the bacterial suspensions, with a microtiter plate. After addition of erythrocytes to each well and incubation of the plates first for 30 min at 37 C and then for 2 h at 4 C, the agglutination titer was determined. Carbohydrate inhibition. PBS containing 25 mg of D-mannose per ml was used parallel to pure PBS in all agglutination and adhesion assays. Agglutination reversed by D-mannose was designated MSHA (mannose-sensitive hemagglutination). MRHA signifies hemagglutination unaffected by D-mannose. Adhesion. Adhesion was tested as described earlier (29). Epithelial cells of squamous and transitional type from the sediment of the urine of one female donor were suspended in PBS. To 105 epithelial cells were added 10' bacteria and PBS with or without D-mannose to a volume of 1 ml. After rotation for 60 min at 370C unattached bacteria were eliminated by repeated washing. The number of bacteria attached to the epithelial cells was counted by direct light microscopy with a Burker chamber. Adhesion is given as the mean number of bacteria attached to 40 epithelial cells. RESULTS Agglutination properties. The capacity to agglutinate erythrocytes or yeast cells (or both) was found in 373 of the 453 E. coli tested. Table 1 shows the number of agglutinating each cell type and the effect of D-mannose on this agglutination. E. COLI IN URINARY TRACT INFECTIONS 565 TABLE 1. Total agglutination pattern of 453 E. coli isolated from urine ofpatients with various forms of UTI or from the stools of healthy children Total aggluti Total Cell type used No. of No. of non-ag- MRHAMSHA nating-glutinating Human eryth rocytes Guinea pig erythrocytes Horse erythro cytes Yeast cells " Cell type used for agglutination in a 3% suspension in PBS with or without 25 mg of D-mannose per ml. Five combinations of hemagglutination patterns could be distinguished (Table 2): (i) inducing only MRHA of human erythrocytes (4% of the 373 with positive agglutination); (ii) causing both MRHA of human and MSHA of guinea pig erythrocytes (37%); (iii) causing MRHA both of human and guinea pig erythrocytes (2 %); (iv) causing MSHA both of guinea pig wnd human erythrocytes (27%); and (v) not agglutinating human erythrocytes but inducing MSHA of guinea pig erythrocytes (30%). The remaining did not agglutinate any of the erythrocytes tested. Agglutination in relation to clinical ongin ofthe. The agglutination properties of the isolated from patients with acute pyelonephritis, acute cystitis, or asymptomatic bacteriuria and from the stools of healthy children are shown in Table 3 and Fig. 1. The MRHA of human erythrocytes was induced by 77% of acute pyelonephritis, 35% of acute cystitis, 18% of asymptomatic bacteriuria, and 16% of normal fecal isolates. The largest number of with only MRHA was found in the pyelonephritis group. Most with MRHA, however, simultaneously induced MSHA (Fig. 1). Strains inducing MSHA of guinea pig erythrocytes were abundant in all diagnosis groups. The highest proportion of with MSHA was found among acute cystitis (91%). The number of without agglutinating capacity was lowest among acute pyelonephritis and highest among the normal fecal isolates. Agglutination in relation to attachment to human urinary tract epithelial cells. The mean adhesion values for the E. coli in relation to their hemagglutination pattern is shown in Table 2. In Table 4 and Fig. 1 the data are grouped after the origin of the. Re-

3 566 'HAGBERG ET AL. TABLE 2. Relation between hemagglutination and attachment to human urinary tract epithelial cells of 453 E. coli Mean adhesion Erythrocyte agglutinated' Meran pesion Group No. of % of b (bacteria per cell) in: Human Guinea pig PBS D-mannose I MR II MR MS III MR MR IV MS MS V - MS VI a MR, Mannose resistant; MS, mannose sensitive; -, no agglutination. 'Percent of with positive hemagglutination (373 out of 453). TABLE 3. Agglutination pattern in relation to origin of 453 E. coli isolated from the urine of patients with various forms of UTI or from the stools of healthy children % of in each diagnosis group agglutinating: Total non- Diagnosis Human erythrocytes Guinea pig erythrocytes agglutinating MRHA MSHA MRHA MSHA Pyelonephritis Cystitis Asymptomatic bacteriuria Fecal gardless of origin, high adhesive capacity was found for with MRHA of human erythrocytes. Thus, 96% attached with 210 bacteria per cell, and 86% attached with -30 bacteria per cell. A significant correlation (r = 0.43) was found between the MRHA titers and the mean adhesion values of the pyelonephritis. The inducing only MSHA had low mean adhesion (Table 4). Thus, 36% attached with -10 bacteria per cell, and 18% attached with 230 bacteria per cell. No correlation was found between the number of positive MSHA steps and the mean adhesion values, tested for acute pyelonephritis. Out of the 94 without capacity to agglutinate erythrocytes and yeast cells, 4 attached with 210 bacteria per cell, and none attached with >30 bacteria per cell. Effects of D-mannose on attachment. The attachment of inducing MRHA of human erythrocytes alone or in combination with MSHA of guinea pig erythrocytes was weakly affected by D-mannose (Tables 2, 4, and 5). The low attachment of with MSHA of guinea pig erythrocytes was more strongly decreased by mannose, especially with from patients with acute cystitis (P < 0.01). Strains inducing MSHA of both human and guinea pig erythrocytes were especially numerous in the acute cystitis group (Table 4). In Table 5 is also confirmed the difference in adhesive capacity described earlier (31) between isolated from INFECT. IMMUN. patients with acute pyelonephritis, acute cystitis or asymptomatic bacteriuria and from the stools of healthy children. DISCUSSION Bacteria entering the urinary tract may induce symptomatic infections involving the kidney as acute pyelonephritis or infections limited to the bladder as acute cystitis. Bacteriuria may also be present in the absence of symptoms (11, 19, 20). The type of UTI resulting in a susceptible host partly depends on the combination of virulence factors of the infecting strain, such as lipopolysaccharide, 0 antigens, capsular polysaccharide, K antigens, resistance to the bactericidal effect of human serum, and ability to attach to human urinary tract epithelial cells. E. coli isolated from patients with acute pyelonephritis often possess all of the virulence factors in combination, which normal fecal isolates very rarely do. In particular, the capacity to attach to vaginal, periurethral, and urinary tract epithelial cells is considered to be a factor determining which of the faecal E. coli may reach and colonize the urinary tract (8, 17, 31). The results of the present study indicate that the bacterial surface antigens responsible for attachment to human urinary tract epithelial cells also induce mannose-resistant agglutination of human erythrocytes. Antigens inducing other types of

4 VOL. 31, 1981 Acute pyelonephritis E. COLI IN URINARY TRACT INFECTIONS 567 Acute cystitis Asymptomatic bacteriuria Normal faecal flora Downloaded from FIG. 1. Relation between hemagglutination and adhesion of E. coli from patients with various forms of UTI or from the stools of healthy children. The sectors within each circle represent the proportion of in the group with the respective hemagglutination pattern. The figure represents the mean adhesion value for all the in the sector (bacteria per cell). Strains'with MRHA alone or together with MSHA showed high mean adhesion regardless of origin. The highest proportion of with MRHA was found among acute pyelonephritis. Most with MRHA simultaneously caused MSHA. NA, Nonagglutinating. hemagglutination are weakly or not at all involved in the binding to urinary tract cells. Hemagglutination may be a simple risk test for urinary E. coli isolates. Hemagglutination has been used to classify enterobacterial adhesins (3, 4). Judging from the hemagglutination pattem, two main classes of adhesins were found on the UTI pathogens examined in the present study. The first class corresponded to type 1 pili, and the second class consisted of the MRHA-inducing adhesins. Two serological types of MRHA adhesins were designated fimbriae 7 and 8 (F7 and F8) by 0rskov et al. (26). Evidence that these adhesins are of a pilus nature has been obtained through work with isolated pili free of detectable contaminating proteins, lipopolysaccharide, or capsular polysaccharide. Pili isolated from three E. coli had the same hemagglutination pattern as whole piliated bacteria (27, Korhonen and Svanborg Eden, submitted for publication), 25Ilabeled pili bound specifically to human urinary tract epithelial cells (15), and antipilus antibodies inhibited attachment (Korhonen and Svanborg Ed6n, submitted for publication). Most previous studies of the role of pili on urinary tract on April 11, 2019 by guest

5 568 HAGBERG ET AL. 42 C.) 0 4) 46). SXi r.t 0. II0 04 4) cog _~ 4) i '04 m.i "Si I. A 9 ' 4) 4 r Z i2.5 r.s is.i 4) l co *R Z i 4.45.i Z r ȯ Z 0 0" -4 1e Nq Co e CO L0 Cq e eq Cq "410 co co co eqcq t- t- X _4 t "4 Co4 0Qc;. *g qr Cq co H eq _- "-4 Cq eq pathogens have not considered that several types of pili with identical morphology may be present simultaneously on the E. coli. Of the causing MRHA in the present study, 85% also induced MSHA (10, 25). Furthermore, nonfimbriate hemagglutinins may be found on enterobacteria, especially on causing MRHA of human erythrocytes (4). Of the 16 causing only MRHA of human erythrocytes, 7 were found to be piliated by electron microscopy (Korhonen et al., unpublished observations). The possible presence of pili on the remaining with MRHA adhesins has not been investigated. The capacity to induce MRHA of various erythrocytes of E. coli isolated from the urinary tract was mentioned by Duguid in 1968 (3). A higher proportion of MRHA of human erythrocytes was among from UTI as compared with normal enteric flora (21, 24, 32), and a relation between MRHA and virulence in the chicken embryo model (24), and a relation between MRHA and adhesion to periurethral cells (16) have been demonstrated. However, none of these studies has considered the type of UTI from which the E. coli were isolated. When differentiating between more or less severe UTI, a strong relation between virulence and MRHA of human erythrocytes became evident, with 77% of acute pyelonephritis and only 16% of normal fecal isolates inducing such hemagglutination. The virulence factor illustrated by MRHA is the capacity to attach to human uroepithelial cells. Virulence related to adhesive capacity and MRHA of human erythrocytes has been described also for colonization factor antigen-carrying E. coli causing diarrhea in humans (6, 7). The mannose-resistant adhesins on urinary tract and intestinal E. coli pathogens have not been shown to cross-react. Due to their specificity, such adhesins may prove suitable as vaccine antigens (2) since the risk of interfering with the ecological balance on other mucous membranes would be small. Of the 453 tested, 349 bound to mannose residues, as shown by yeast cell agglutination (23). A mannose-containing receptor is unlikely to be important on human uroepithelial cells, since with MSHA alone attached in low numbers or not at all to these cells. Urinary slime or Tamm-Horsfall protein is rich in mannose residues. Recently 0rskov et al. described binding to urinary slime mediated by type 1 pili (26). Facilitated colonization of mucus-covered microbal surfaces might explain the high frequency of type 1 pili on most enterobacteria (3). Binding to urinary slime may have a dual effect by increasing the excretion of bacte- co t'- "4 CO -4 _-"4 I,- 0CD 4 CO "-4 S '4 I.0 VP4 E-q 0 4) 4) 14) 0: 5.0 ~4).Sz. INFECT. IMMUN.

6 VOL. 31, 1981 E. COLI IN URINARY TRACT INFECTIONS 569 Effect of D-mannose on the adhesion to human urinary tract epithelial cells of E. coli from TABLE 5. various forms of UTI Mean adhesion % Adhering with >10 bacteria Diagnosis No. of (bacteria per cell) in: per cell in: Saline D-Mannose Saline D-Mannose Pyelonephritis Cystitis Asymptomatic bacteriuria Fecal ria bound to slime or allowing bacteria to remain in the bladder associated with slime rather than attached to the epithelium or both. To induce infection bacteria may need to penetrate the mucous layer and attach to the epithelial cells. The with MRHA adesins bind to glycolipid receptors on the human uroepithelial cells and erythrocytes (18). One receptor is shared on globoseries glycolipids. Globotetraosylceramide efficiently inhibits bacterial attachment, and agglutination ofunreactive cells may be induced after coating with globoside (18, 34). Over 80% of the with MRHA adhesins reported here bind to globoside (Leffler and Svanborg-Eden, manuscript in preparation). The majority of these, however, also form type 1 pili. Competitive binding to slime or to glycolipids in the epithelium may determine the level and tissue engagement of UTI. Agents blocking the tissue receptors may prove efficient in prophylaxis against infection. ACKNOWLEDGMENTS We greatly appreciate the technical assistance of Lise-Lotte Johansson and Helen Akesson. This study was supported by grant 215 from the Swedish Medical Research Council, grant EKB-U-614 from the Swedish Board for Technical Development, and grants from the Medical Faculty, University of Goteborg, and the Ellen, Walter and Lennart Hesselman Foundation for Scientific Research. LITERATURE CITED 1. Brinton, C. C., Jr The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N. Y. Acad. Sci. 27: Brinton, C. C., Jr The piliation phase syndrome and the uses of purified pili in disease control, p XIII U.S.-Japan Conference on Cholera. Publisher, City of Publication. 3. Duguid, J. P The function of bacterial fimbriae. Arch. Immunol. Ther. Exp. 16: Duguid, J. P., S. Clegg, and M. L. Wilson The fimbrial and nonfimbrial hemagglutinins of Escherichia coli. J. Med. Microbiol. 12: Duguid, J. P., and R. R. Gilies Fimbriae and adhesive properties in dysentery bacilli. J. Pathol. Bacteriol. 74: Evans, D. J., Jr., D. G. Evans, and H. DuPont Haemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with hu' man, bovine, chicken, and guinea pig erythrocytes in the presence and absence of mannose. Infect. Immun. 23: Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G. Chase, and S. L. Gorbach Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect. Immun. 12: Fowler, J. E., and T. A. Stamey Studies of introital colonization in women with recurrent urinary tract infection. VII. The role of bacterial adherence. J. Urol. 117: Gibbons, R. J Bacterial adherence in infection and immunity. Rev. Microbiol. (Sao Paulo) 4: Gupta, N. P., S. P. Gupta, and D. Barua Haemagglutination by of Escherichia coli isolated from cases with urinary infection. Indian J. Med. Res. 46: Hanson, L. A., S. Ahlstedt, A. Fasth, U. Jodal, B. Kaijser, A. Sohl Akerlund, and C. Svanborg Eden Antigens of E. coli, human immune response and the pathogenesis of urinary tract infections. J. Infect. Dis. 136(Suppl.): Houvink, A. L, and W. van Iterson Electron microscopical observations on bacterial cytology. II. A study on flagellation. Biochim. Biophys. Acta 5: Jodal, U., U. Lindberg, and K. Lincoln Level diagnosis of symptomatic urinary tract infections in childhood. Acta Paediatr. Scand. 64: Korhonen, T. K Yeast cell agglutination by purified enterobacteria pili. FEMS Microbiol. Lett. 6: Korhonen, T. K., S. Eden, and C. Svanborg Eden Binding of purified Escherichia coli pili to human urinary tract epithelial cells. FEMS Microbiol. Lett. 7: Kallenius, G., and R. Moilby Adhesion of Escherichia coli to human periurethral cells correlated to mannose-resistant agglutination of human erythrocytes. FEMS Microbiol. Lett. 5: Kililenius, G., and J. Winberg Bacterial adherence to periurethral epithelial cells in girls prone to urinary tract infections. Lancet ii: Leffler, H., and C. Svanborg Eden Chemical identification of a glycosphingolipid receptor for Escherichia coli attaching to human urinary tract epithelial cells and agglutinating human erythrocytes. FEMS Microbiol. Lett. 8: Lidin-Janson, G., L. A. Hanson, B. Kaijser, K. Lincoln, U. Lindberg, S. Olling, and H. Wedel Comparison of Escherichia coli from bacteriuric patients with those from faeces of healthy school children. J. Infect. Dis. 136: Lindberg, U., L. A. Hanson, U. Jodal, G. Lidin-Janson, K. Lincoln, and S. Olling Asymptomatic bacteriuria in schoolgirls. II. Differences in E. coli caus-

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