Regulation of Production of Type 1 Pili among Urinary Tract Isolates of Escherichia coli

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1 INFECTION AND IMMUNITY, Dec. 1986, p Vol. 54, No /86/ $02.00/0 Copyright 1986, American Society for Microbiology Regulation of Production of Type 1 Pili among Urinary Tract Isolates of Escherichia coli SCOTT J. HULTGREN,1 WILLIAM R. SCHWAN,2 ANTHONY J. SCHAEFFER,2 AND JAMES L. DUNCAN'* Departments of Microbiology-Immunologyl and Urology,2 Northwestern University Medical School, Chicago, Illinois Received 14 April 1986/Accepted 21 August 1986 The piliation and hemagglutination properties of 54 consecutive Escherichia coli isolates from women with recurrent urinary tract infections were studied. Mannose-sensitive hemagglutination (MSHA) of guinea pig erythrocytes, characteristic of type 1-piliated bacteria, was produced by 75% of the isolates, 32% produced mannose-insensitive hemagglutination, and 14% produced no hemagglutination reaction. The production of type 1 pili was examined in those strains that produced MSHA only. Studies with antiserum prepared against purified pili suggested that at least three subtypes of type 1 hemagglutinins were represented among the isolates. All of the type 1-piliated isolates produced MSHA after serial subculture in static broth. After growth on agar, selected type 1-piliated isolates were subdivided into two groups. Many strains apparently suppressed piliation during growth on agar (regulated variants); all colonies became MSHA negative and were composed of nonpiliated cells as shown by electron microscopy. The loss of the MSHA phenotype often occurred after a single overnight passage on agar, and any remaining hemagglutinin was gradually lost with one to three additional passages. Seven strains, however, retained a signfficant hemagglutination titer after multiple subcultures on agar, and they produced colonies consisting of a mixed population of piliated and nonpiliated cells. These strains were apparently able to oscillate between states of pilus expression and nonexpression during growth on agar (random phase variants). When nonpiliated cells isolated from the mixed, random variant population were plated on agar, they gave rise to hemagglutination-positive colonies that consisted of both piliated and nonpiliated cells. The distinction between random variants and regulated variants was also observed in shaking broth cultures inoculated with nonpiliated cells. The random variants produced MSHA-positive cultures composed of piliated and nonpiliated cells, whereas the regulated strains remained nonpiliated. The results indicate that type 1 pili are a predominant adhesin of uropathogenic E. coli and that during growth on agar only about one-fourth of the type 1-piliated isolates regulate pilus expression by random phase variation. Type 1 pili (fimbriae) are filamentous appendages found on the surface of Escherichia coli and other gram-negative bacteria (4, 11). These hairlike structures appear to mediate the hemagglutination (HA) that occurs when piliated bacteria are mixed with erythrocytes of certain species (9, 12). The reaction is inhibited by mannose, and mannose-sensitive hemagglutination (MSHA) of guinea pig erythrocytes has become a functional test for the presence of type 1 pili (8, 11, 30). Type 1 pili are also thought to be responsible for the mannose-sensitive adherence of bacteria to other types of eucaryotic cells, a process considered to be an important initial step in certain gram-negative infections (8, 19, 25, 31). E. coli is the most common cause of urinary tract infections, and type 1 pili may allow this organism to adhere to uroepithelial cells (1, 32, 36). Most E. coli isolates produce type 1 pili (6, 11, 27), and a number of experiments in animal models of infection have provided evidence which suggests that type 1 pili are important in colonization of the urinary tract mucosa (1, 3, 17, 34). The production of type 1 pili by E. coli is affected by phase variation, a random on-off switching process that allows the cells to alternate between piliated and nonpiliated states at rates estimated to be 10-2 to 10-3 per cell per generation (4, 14, 20, 35). Studies with pil-lac operon fusions in E. coli K-12 strains have suggested that phase variation is the consequence of metastable transcriptional control of the pilus subunit structural gene, pila (14, 29). It has recently been * Corresponding author. 613 shown that genome rearrangements occur during the switching process and that the pilus on-off switch may be mediated by the inversion of a 314-base-pair segment of DNA immediately upstream of pila (2). In addition to random variation of pilus expression, the production of type 1 pili is known to be affected by the conditions of culture (8, 12, 13). For example, subculture in static broth results in a bacterial population that is heavily piliated, whereas growth of most strains on agar produces bacteria with few or no pili (11). The relationship between random phase variation and the effects of growth conditions on piliation is uncertain. Most of the experiments describing phase variation have made use of derivatives of laboratory E. coli B and K-12 strains. In the work described here, we have examined piliation in 54 consecutive clinical isolates of E. coli from women with urinary tract infections and characterized pilus production in the type 1-piliated strains. Selected isolates could be divided into two groups based on their piliation and HA properties after growth on agar. The majority of the strains appeared to completely suppress pilus production on agar. Other isolates, however, continued to produce pili when grown on agar in a manner characteristic of random phase variation. MATERIALS AND METHODS Patient population. The patients were adult women with urinary tract infections who were seen as outpatients in a urology clinic. Urine specimens were obtained by urethral catheterization and cultured as described below. The popu-

2 614 HULTGREN ET AL. lation consisted of one patient with no previous urinary tract infection and 53 patients with recurrent infections, 34 of whom had two or more infections in the preceding year. Sixteen patients had significant urinary tract abnormalities including chronic pyelonephritis (3 patients), infection stone (1 patient), infected ureteral stump (1 patient), and residual urine (11 patients). Thirty-eight women with acute symptoms were judged to have infection limited to the lower urinary tract. Based on additional clinical findings, four women were judged to have acute pyelonephritis. Twelve women with documented infection were asymptomatic. Bacteria. Fifty-four consecutive isolates of E. coli were cultured on MacConkey and blood agar plates; greater than 105 organisms were present in each specimen. Those isolates that were identified as E. coli on the basis of biotyping were stored as frozen brain heart infusion (BHI) cultures at -70 C, as lyophilized BHI cultures, and on BHI agar slants. For experimental purposes, the isolates were grown in BHI broth, Luria broth (LB), or minimal salts-glucose (MSG) medium [0.05 M K2HPO4, 0.03 M KH2PO4, M (NH4)2SO4, 0.2 mm MgSO4, M sodium citrate, 0.01 M glucose]. Agar passage was usually on MacConkey agar, but in some cases blood or glucose-minimal salts agar plates were used. In some experiments the previously characterized strain 149 (17) was compared with the 54 isolates. HA reactions. HA of a 1% guinea pig or human erythrocyte suspension in phosphate-buffered saline (PBS) was determined for broth-grown organisms by suspending the bacteria to an optical density at 540 nm of 0.80 (4 x 109 to 8 x 109 bacteria per ml), centrifuging a 1-ml amount in an Eppendorf microfuge, and then suspending the pellet in 0.1 ml of PBS. A 25-,I amount (1 x 109 to 2 x 109 bacteria) was serially diluted in microtiter plates containing 25,ul of PBS in each well. An equal volume of erythrocytes was added; after mixing, the plates were incubated at 4 C for 4 to 18 h. The HA endpoint was defined as the last dilution well before erythrocyte buttons were formed. The titers were expressed as the reciprocal of the endpoint dilution. Colonies (50 to 100) of agar-grown organisms to be tested were removed from plates with sterile swabs and suspended to an optical density at 540 nm of 0.94 (7 x 109 to 1 x 1010 bacteria per ml). After centrifugation and suspension, HA titers were determined as described above. In this manner, the HA assay for broth- and agar-grown cultures was standardized so that approximately the same number of bacteria were titered for each strain. An HA titer probably reflects the degree to which indiyidual cells are piliated or the proportion of piliated cells in a population or both. The sensitivity of the HA assay used here is shown by the finding that a ratio of one HA' cell to 500 HA- cells is sufficient to produce a positive HA reaction (unpublished data). The effect of mannose on HA was assessed by incubating the bacteria in PBS containing 1% mannose for 10 min at room temperature before diluting the cells. Strains producing mannose-insensitive HA of human erythrocytes were examined for the presence of P agglutinins by testing their ability to agglutinate human P-positive and p (Pnegative) erythrocytes (18). HA inhibition. Antiserum against pili isolated and purified from E. coli strains 149, 21, and Bam was prepared as described previously (17). The HA titer of the strains to be tested was determined as described above, and the cells were diluted so that each strain gave a titer of 16. The antisera were serially diluted in microtiter plates, and 25,ul of bacterial suspension was added to each well. After INFECT. IMMUN. incubation for 30 min at room temperature, erythrocytes were added, and the plates were incubated at 4 C. The endpoint was defined as the greatest dilution of antiserum that prevented a positive HA reaction. Separation of HA-negative cells. HA-negative cells were separated from HA-positive cells in mixed populations of random phase variants by adsorption to erythrocytes as described by Nowicki et al. (21). Bacteria obtained after growth on agar plates were resuspended to an optical density at 540 nm of 0.80, and 1 ml was incubated with 1.5 ml of a 1% guinea pig erythrocyte suspension for 5 min at room temperature. After a 5-min slow-speed centrifugation, 1 ml of supernatant was collected and concentrated by centrifugation in a Eppendorf microfuge, and the pellet was suspended in 0.1 ml of PBS. These cells were again treated with erythrocytes, and the separation procedure was repeated. The resulting bacterial suspension, which was enriched for HA-negative cells, was diluted and used to inoculate agar or shaking broth cultures. EM. Cells from selected isolates that had been transferred on blood agar for at least five consecutive passages were suspended in 0.1 M cacodylate buffer (ph 7.3) and prepared for electron microscopy (EM) as previously described (17). RESULTS HA reactions. The HA reactions of 54 consecutive E. coli isolates from women with urinary tract infections plus two colony morphology variants were determined after -5 consecutive passages in static broth or on agar. MSHA of guinea pig erythrocytes indicative of type 1 pili was produced by 75% of the isolates (groups I and II); 54% produced only this reaction (Table 1). Mannose-resistant HA of human or guinea pig erythrocytes was produced by 32% of the strains, with 11% producing the mannose-resistant HA reaction only. The isolates that produced mannose-resistant HA of human cells were tested further to determine whether agglutination involved P blood group antigens. Of the 19 strains tested, 16 gave a positive reaction with human P erythrocytes and a negative reaction with p erythrocytes, indicating the presence of P hemagglutinins (26). Eight isolates (14%) did not agglutinate guinea pig or human cells. Four of the 54 isolates exhibited smooth-rough variation in colony morphology when plated on agar; the rough colony variants gave rise to predominately smooth colonies after passage in static broth. For two of the four isolates (2-23 and 2-24) the HA pattern remained the same for both the smooth and rough phenotypes. Variants of strains 2-11 and 2-22, however, gave rise to different HA patterns (Table 1). Although all of the strains producing MSHA in group I of Table 1 that were examined by EM possessed pili after growth in broth, it appears that the type 1 pili are not identical. For example, only 8 of the 30 strains gave a definite reaction with human erythrocytes. To further document the heterogeneity of type 1 pili, several of the isolates were examined in an HA inhibition test with antibody prepared against purified type 1 pili of three antigenically distinct subtypes (strains 149, Bam, and 21). Broth cultures of the strains to be tested were diluted to give an HA titer of 16, and samples of the cells were added to microtiter plate wells containing serial dilutions of antibody. After the addition of erythrocytes, the plates were incubated at 4 C, and the greatest antibody dilution which inhibited agglutination was noted. Several isolates produced type 1 pili that appeared to be more closely related to the type 1 pili of strain 149, others were more related to strain 21 pili, and the pili of

3 VOL. 54, 1986 TABLE 1. HA reactions of 54 consecutive E. coli urinary tract isolates HA reactiona Isolate Guinea pig Human erythrocytes erythrocytes Broth Agar Broth Agar Group I 2-3 (32),b 2-8 (512), 2-13 (12), (2,048), 2-17 (2,048), 2-19 (16), 2-21 (1,024), 2-22rc (64), 2-23 (512), 2-25 (512), 2-27 (4), 2-29 (1,024), 2-35 (64), 2-36 (128), 2-38 (512), 2-39 (1,024), 2-43 (16), 2-45 (256), 2-48 (8), 2-56 (64) 2-24 (2,048), 2-53 (1,024), 2-57 (512) (1,024), 2-40 (64) (4,096), 2-9 (2,048), 2-14 (2,048), (256) 2-5 (4,096) Group II 2-16, 2-34, 2-49, 2-52, R R 2-1, 2-28, R R + R /R + +/R R /R + + R /R R - - Group III 2-12, 2-31, R R 2-4, 2-6 R - R R 2-llr R R R R Group IV 2-2, 2-10, 2-18, 2-22, 2-26, 2-41, 2-44, a +, MSHA; -, no reaction; R, mannose-insensitive HA; +/R, HA that was only partially inhibited by mannose. b Numbers within parentheses indicate the highest titers obtained in static LB or BHI broth culture. c r, Rough colony variant. one isolate were most closely related to the type 1 pili produced by strain Bam (Fig. 1). Some isolates (e.g., 2-8 and 2-9) gave only a minimal reaction with all three test sera. The results indicate that the type 1 pili produced by these isolates are antigenically diverse and that at least three and probably more subtypes of type 1 pili are represented. An additional difference in the type 1-piliated organisms 10 lo -06 I-K STRAIN FIG. 1. HA inhibition of E. coli isolates by antipilus antiserum. Antibody to pili of strains 149 (LI), Barn (U), and 21 (U). REGULATION OF TYPE 1 PILUS PRODUCTION 615 w CP: z 0 z -J w NUMBER OF PASSAGES FIG. 2. Loss of MSHA after passage of broth-grown E. coli strains on blood agar. Strains: 2-16 (0), 2-35 (0), 2-38 (A), 2-1 (A); 2-36 (-). can be seen in Table 1. Most of the isolates lost the ability to produce MSHA after five consecutive passages on blood agar, but a few isolates remained MSHA positive after continuous subculture on agar. This difference in type 1 pilus production was examined in more detail. Production of mannose-sensitive hemagglutinins in broth and on agar. All of the strains in groups I and II of Table 1 were MSHA positive for guinea pig erythrocytes (type 1 piliated) after serial subculture in static broth, but the majority became MSHA negative after passage on agar plates. This was determined by examining cells from hundreds of colonies of each strain in the HA assay. To further demonstrate that these strains had become HA negative, 103 individual colonies of a representative strain, 149, were picked to separate wells of microtiter plates and tested for HA. Every colony tested was HA negative. The kinetics of the process whereby certain strains became HA negative was studied by following the decline in HA titer during successive passages on agar. The HA titer of broth-grown cells was dramatically reduced or eliminated by a single overnight passage on agar (Fig. 2), and any remaining hemagglutinin was gradually lost during the subsequent one to three agar passages. Strain TABLE 2. Conversion of HA-negative isolates to an HA-positive state HA- HA' No. of passagesa 5 Total h a In static cultures of BHI broth.

4 616 HULTGREN ET AL. P- -7 z 6-0,; 2 z STRAIN FIG. 3. HA of selected E. coli isolates after -5 passages on agar. Strains that had become HA negative on agar were inoculated into broth and tested for the reappearance of mannose-sensitive hemagglutinins after growth in static cultures at 18, 24, 48, and 72 h and at 24, 48, and 72 h of the second and third subcultures. The number of subcultures and the total time required for MSHA-negative cultures to become positive (HA titer of 3 to 8) for selected strains are shown in Table 2. Although approximately 50% of the strains became positive in the first subculture, considerable variation was observed among the different isolates. Additional experiments revealed that the reappearance of the MSHA reaction depended on the number of subcultures and not simply on the total number of hours in broth. For example, strain 2-17 became MSHA positive after 24 h of the third subculture (168 total h) but remained negative after 168 h in the first subculture tube. In contrast to the strains described above that became HA negative after serial subculture on agar, seven strains retained a significant although reduced HA titer after -5 subcultures on agar (Fig. 3). Individual colonies from these strains were always HA positive, and when 103 individual colonies from a representative strain, 2-7, were picked and tested for HA, every colony was found to be HA positive. Three such strains have been passaged more than 50 times on agar and remain HA positive. EM. EM examination of the strains in group I of Table 1 revealed that some strains which became MSHA negative after subculture on agar continued to produce pili. These may be nonfunctional type 1 pili (e.g., type 1C) or other unrelated pili that do not mediate HA of guinea pig or human erythrocytes. In other strains, however, it was possible to unambiguously show a correlation between pilus production and MSHA. Examination of 50 to 100 cells of isolates in this group revealed that those strains that became MSHA negative on agar consisted entirely of cells that were devoid of pili (Table 3). However, the strains that remained MSHA positive on agar consisted of a mixed population of cells (Table 3; Fig. 4); a large proportion of the cells was nonpiliated or possessed very few pili, and a similarly large proportion was heavily piliated. The HA and EM results suggested that the type 1-piliated isolates could be subdivided into two groups based on differences in pilus production. The largest group consisted of strains that produced pili in static broth cultures but became completely nonpiliated after serial subculture on agar (regulated variants). The second group also produced pili in static broth, but after subculture on agar, these strains INFECT. IMMUN. gave rise to HA-positive colonies composed of a mixed population of piliated and nonpiliated cells (random variants). The random variants were studied further by isolating nonhemagglutinating cells from the mixed population. The cells of random variant strains growing on agar were suspended in broth and then incubated with an excess of guinea pig erythrocytes to remove the HA-positive bacteria. This process was repeated, and the resultant nonadherent cells were diluted and plated on blood agar. The colonies that arose were then tested and for each strain; 10 of 10 colonies were HA positive. EM revealed that each colony consisted of both piliated and nonpiliated cells. These results show that nonhemagglutinating cells present in the random variant population can give rise to HA-positive cells and suggest that during growth, a random on-off switching process leads to the development of colonies consisting of a mixed population of piliated and nonpiliated cells. Production of pili in shaking broth cultures. The regulated and random variant strains cannot be distinguished in static broth cultures. Under those conditions, the cells of both groups become predominantly piliated, perhaps due to the selective overgrowth of piliated cells (1, 28). When HAnegative cells isolated as described above from random variant strains were inoculated into shaking LB or BHI broth, the cultures became HA positive (Table 4) and consisted of a mixture of piliated and nonpiliated cells. In contrast, however, HA-negative cells of agar-grown regulated variants remained HA negative and nonpiliated after growth in shaking LB or BHI broth, except for strain 2-45, which produced a titer of 4. When the same regulated variant strains were grown in a shaking culture of MSG medium, four of the strains produced MSHA titers; the same four strains also became HA positive on blood agar plates incubated anaerobically and on MSG agar plates incubated aerobically. These results confirm that nutritional and environmental factors are important in the regulation of pilus production by some strains. Pilus production by E. coli Bam. A number of investigators have observed distinct colony morphology differences with certain laboratory E. coli K-12 strains which correlated with production of type 1 pili (4, 30, 35). Large, flat colonies with ragged edges contained nonpiliated cells, whereas raised, smooth colonies contained piliated cells. We compared pilus production by the clinical isolate 149 with the laboratory strain Bam to provide insight into the different regulatory properties that govern piliation in these two strains. After TABLE 3. EM examination of selected isolates after.5 subcultures on agar No. of cells with the following no. of Strain HA pili per titer cell: > , 2-23, 2-25, 2-36, , 2-39, 2-45, 2-56, r

5 VOL. 54, 1986 REGULATION OF TYPE 1 PILUS PRODUCTION 617 Downloaded from FIG. 4. growth in static broth, Bam was plated on blood agar, and an isolated raised, smooth colony and a flat, rough colony were picked and restreaked. Typical smooth and rough colonies were transferred on agar in this way through a total of 10 passages, at which time one culture consisted of predominantly raised, smooth HA-positive colonies with a culture HA titer of 128. The other culture consisted entirely of large, flat, ragged colonies which were HA negative; overall, the culture had an HA titer of 2. Thus, with strain Bam, the colonial phenotypes could be passaged and separately maintained on agar. With the clinical strain 149 (which shows no colony morphology differences) HA-positive colonies were selected by the slide test for subsequent passage on agar. However, after three passages on agar only 10% of the colonies tested remained weakly HA positive, and by the fourth passage piliation had been completely suppressed; no HA-positive colonies could be detected, the culture had no titer, and all of the bacteria were devoid of pili as determined by EM. Thus, with strain 149, which is typical of the regulated variants we have studied, it was not possible to selectively maintain piliated colonies on agar. The results suggest that the regulation of pilus production by laboratory Cells from a random variant colony (2-33) after 25 passages on agar (x48,700). strains of E. coli may be fundamentally different from that of recent clinical isolates. DISCUSSION The occurrence of phase variation of type 1 pili in E. coli has been known for many years. Brinton originally described the on-off switching process in E. coli laboratory strains (4, 5), and recent evidence indicates that phase variation is the result of random inversion of a 314-base-pair segment of DNA upstream of the pilus structural gene (2). The work of Duguid and co-workers (8, 10, 11) has documented the effects that different environmental growth conditions have on type 1 pilus production, but it is not clear how these effects, many of which seem regulatory or selective (or both) in nature, are related to random phase variation. Our examination of 54 clinical isolates of E. coli from the urinary tract indicates that most type 1-piliated strains are able to suppress pilus production during growth on complex agar medium as well as under certain other growth conditions. About one-fourth of the type 1-piliated isolates, however, expressed pili by a random phase variation process which on March 11, 2019 by guest

6 618 HULTGREN ET AL. TABLE 4. Effect of growth conditions on type 1 pilus production by nonpiliated cellsa HA titer Strain Statis 5Xb Blood 5X Shaking 4X Shaking LB agar LB MSG Regulated variants , , , Random variants 2-7 -c *d * * * * * * 16 - a Nonpiliated cells of the regulated strains were taken directly from blood agar plates. Nonpiliated cells were isolated from random variant strains by differential adsorption to guinea pig erythrocytes. b X, Number of passages. c, Not determined. 10 of 10 colonies tested were HA positive. d *, resulted in the production of both piliated and nonpiliated cells under all growth conditions tested. All of the isolates capable of producing type 1 pili did so after subculture in static LB or BHI broth. When these organisms were then subcultured on blood agar or Mac- Conkey agar, two modes of pilus expression could be distinguished. Those strains that we describe as regulated variants became HA negative after passage on agar, often after a single subculture. Every colony tested proved to be HA negative, and EM showed the cells to be devoid of pili. The absence of pili was confirmed for the culture as well as for individual colonies. In contrast to the regulated variants, a small group of strains continued to produce positive HA reactions after multiple subcultures on agar. The HA titer produced by these strains on agar was less than that produced in static broth, perhaps because of selective outgrowth of piliated cells in the latter situation (1, 28) but nevertheless was significant, ranging from 16 to 256. When we examined individual colonies, rather than finding a mixture of HApositive and -negative colonies, we found that every colony produced a HA-positive reaction; EM of individual colonies showed that they consisted of a mixture of piliated and nonpiliated cells. These strains appear to be random variants, capable of randomly oscillating between states of pilus expression and nonexpression during growth on agar. Pilus production in the regulated variants, in contrast, appears to be suppressed in every cell when growing on agar. To show that the HA-negative cells in the random variant population could give rise to both piliated and nonpiliated cells, we isolated nonpiliated cells by erythrocyte absorption techniques and then cultured those cells on agar. Every colony tested that arose from the HA-negative inoculum was HA positive and consisted of a mixture of piliated and nonpiliated cells. Our results with the random phase variants confirm and INFECT. IMMUN. extend those of Nowicki et al., who have made use of antibodies tagged with photofluors to study phase variation of type 1 and other pili (20, 22). They observed a high rate of phase variation (10-2 per cell generation) and also found that the colonies of phase variants consisted of mixtures of piliated and nonpiliated cells (23). The relationship of our results to those of Eisenstein (14) and Orndorff et al. (29), who examined phase variation in pil-lac operon fusion strains, is less clear. Those investigators described the presence of separate Lac' and Lac- colonies on appropriate lactose-containing medium. If one assumes that random phase variation occurs during growth on agar and that the rate is 10-2 to 10-3 per cell per generation, every colony should be a mixture of piliated and nonpiliated cells. With an inoculum of nonpiliated cells, for example, it can be calculated that between 106 and 5 x 107 piliated cells would be generated in a colony of 108 cells at the lower rate of phase variation. These ratios of piliated cells to nonpiliated cells in a colony would easily be detected by HA with the procedures described here (unpublished data). In addition to growth on agar, growth in shaking cultures of LB and BHI broth distinguished random variants from regulated strains. When nonpiliated cells from random variant cultures were inoculated into shaking broth, HA-positive cultures arose which consisted of a mixture of piliated and nonpiliated cells. When HA-negative regulated variant cells were grown in shaking LB, however, they continued to produce HA-negative cultures. In parallel to the situation seen on agar, growth of random variants in shaking broth led to a mixed population of piliated and nonpiliated cells, presumably the result of the rapid rate of phase variation. Pilus expression by the regulated strains, however, appeared again to be suppressed in shaking LB cultures. The possibility that nonpiliated cells were outgrowing piliated cells in the latter situation seems unlikely and was ruled out by showing that piliated and nonpiliated cells grew at the same rate in shaking broth cultures (data not shown). The results discussed above indicate how sensitive pilus expression is to changes in the environmental or nutritional conditions of growth. Nonpiliated cells of regulated variants became piliated during growth in static LB, but remained nonpiliated in the same medium under shaking conditions. The composition of the medium also seems to be important because four of the nine regulated variant strains were able to produce type 1 pili when nonpiliated cells were grown in shaking cultures of a MSG medium (Table 4). The cells in these cultures consisted of both piliated and nonpiliated cells, suggesting that at least some (perhaps all) of the regulated variants may be able to randomly phase vary the expression of pili under certain conditions. In contrast to the regulated variants, the strains which randomly vary pilus production on agar appear to be able to do so under a variety of growth conditions. Evidence for the heterogeneity of type 1 pili was obtained from the differential HA of human erythrocytes by piliated isolates (Table 1; for an alternative explanation, see reference 24), and more directly by the use of antisera prepared against purified pili (Fig. 1). Other investigators have described the heterogeneity of type 1 pili (15, 33), and our results suggest that at least three and probably several more subtypes of type 1 pili are represented in the clinical isolates we examined. The results also show that in contrast to certain laboratory K-12 strains (30, 35), there appears to be very little correlation between colony morphology and type 1 piliation in clinical isolates of E. coli. Four of our 54 isolates showed differences in colony morphology, but in

7 VOL. 54, 1986 only two of these strains was this difference related to pilus production (Table 1). The E. coli laboratory strain Bam produces piliated and nonpiliated colonial phenotypes, both of which can be selectively passaged and separately maintained on agar. This situation is contrary to the behavior of most clinical isolates on agar. A piliated, HA-positive phenotype cannot be maintained during passage of regulated variants on complex agar media, presumably because growth on agar triggers the suppression of piliation in those strains. Random variants do not produce HA-negative colonies, presumably due to the rapid rate of phase variation. The regulation of piliation in clinical isolates during growth on agar appears to be considerably different from that of laboratory strains. In addition, colony morphology is not useful as an indicator of piliation in clinical isolates of E. coli. These data confirm that type 1 pili are the most prevalent adhesin associated with uropathogenic bacteria and support the concept that they are an important virulence factor in ascending urinary tract infections (1, 3, 17, 34). We did not identify a relationship between the HA profile and the clinical virulence of these uropathogenic strains. Strains obtained from patients with clinical pyelonephritis, cystitis, or asymptomatic bacteriuria showed various combinations of MSHA, mannose-resistant HA, and no HA. It is now clearly understood that patient symptoms are not reliable for localizing the site or intensity of bacterial colonization within the urinary tract (7). Furthermore, the adhesive characteristics of a strain that are determined by growth in vitro do not necessarily correlate with their expression or significance in vivo (16, 26). Careful assessment of the clinical importance of type 1 and mannose-resistant pili will require documentation of their expression in vivo in animal and human urinary tract infections. ACKNOWLEDGMENTS We thank Louise J. Keating, Northern Ohio Region Red Cross, Cleveland, for providing human p erythrocytes, Margaret Swenson for technical assistance, and Sue Amundsen and Malcolm Winkler for helpful discussions. This work was supported by Public Health Service grant Al from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Abraham, S. N., J. P. Babu, C. S. Giainpapa, D. L. Hasty, W. A. Simpson, and E. H. Beachey Protection against Escherichia coli-induced urinary tract infections with hybridoma antibodies directed against type 1 fimbriae or complementary D-mannose receptors. Infect. Immun. 48: Abraham, J. M., C. S. Freitag, J. R. Clements, and B. I. Eisenstein An invertible element of DNA controls phase variation of type 1 fimbriae in Escherichia coli. Proc. 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