INFECTION AND IMMUNITY, Mar. 1979, p. 690-699 0019-9567/79/03-0690/10$02.

00/0

Vol. 23, No. 3

Hemagglutination and Adhesiveness of Toxigenic Escherichia coli Isolated from Humans

GRACE M. THORNE,* CARL F. DENEKE, AND SHERWOOD L. GORBACH Infectious Disease Service, Department ofMedicine, Tufts-New England Medical Center Hospital, Boston, Massachusetts 02111
Received for publication 15 December 1978

Toxigenic strains of Escherichia coli isolated from humans were studied for adherence to human buccal mucosal epithelial cells. The E. coli strains were labeled with 3H-amino acids or fluorescein isothiocyanate. Toxigenic E. coli strains varied in their ability to adhere in the presence of mannose. Of 32 toxigenic strains examined, 52% bound to the buccal cells, whereas none of 8 control strains did so (Mann-Whitney U test, P = 0.007). The control strains were nontoxigenic E. coli isolates from humans, enterotoxigenic E. coli isolates from animals, and E. coli K-12 containing the K88 or K99 plasmid; these strains exhibited only background-level adherence in this assay. Among the toxigenic E. coli strains that bound to human buccal mucosal cells, there was no correlation with mannoseresistant hemagglutination (MR-HA) of guinea pig and human erythrocytes. Screening 32 strains, we found the following phenotypes: (i) MR-HA', buccal adherent; (ii) MR-HA', buccal nonadherent; (iii) MR-HA-, buccal adherent. Presumably the third group represents strains with another type(s) of surface attachment components not involved in the MR-HA reaction. Our findings indicate that a number of bacterial surface structures can function in MR-HA and buccal adherence.
A recognized virulence factor in enterotoxigenic strains of Escherichia coli is the ability to colonize the upper intestinal tract of humans and animals. As demonstrated by fluorescent antibody, the organisms are- located on the surface of the epithelium, but they do not penetrate the mucosal surface, nor do they cause any perceptible inflammatory response. Since the small bowel is generally free from coliforms in the healthy state, it might be speculated that enterotoxigenic E. coli strains are endowed with a mechanism for overcoming those forces, either chemical or physical, which normally eliminate bacterial contamination from that site. Studies involving E. coli strains which cause diarrhea in animals have provided extensive data concerning this facet of E. coli pathogenesis. In 1961, 0rskov et al. demonstrated the existence of heat-labile K antigens on E. coli isolated from diseased swine (29). This observation was followed by an elegant series of investigations in which the K88 antigen of E. coli was characterized. Briefly, the K88 antigen is a short, fragile, pilus-like structure which projects out from the cell "like fur"; it consists of protein and it is highly antigenic (26, 30, 41, 42). The role of the K88 antigen as a virulence factor was demonstrated in 1971 by Smith and Linggood, who described its mechanism of action as
690

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adhesiveness for the small bowel (38). The K88 antigen was found to be associated with a specific transferable plasmid which proved to be relatively unstable; that is, it was spontaneously lost by the E. coli isolates after subculture in the laboratory (15, 26, 37, 38). Concurrently, it was observed that E. coli enteropathogenic for cows and for lambs possessed a different surface-associated, plasmid-mediated antigen, labeled K99. This antigen was found to confer virulence for cows and lambs, but not for pigs, thus introducing evidence for a mechanism involving host specificity (38, 46). Later work by these and other investigators confirmed earlier observations and strengthened the idea that the K88 and K99 antigens were essential virulence factors for animal-associated E. coli diarrhea (17, 39). Similarly in humans, intestinal colonization by enterotoxigenic E. coli is a necessary prerequisite in acute enterotoxic diarrhea. Studies of E. coli strain H10407 employing an infant rabbit colonization model have implicated a surface antigen termed "colonization factor antigen" (CFA) which has a fimbriate morphology but differs antigenically from K88. CFA may also be controlled by a bacterial plasmid although synthesis of the CFA following plasmid transfer to a recipient has not been accomplished (11).

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There are relatively few studies of the interaction between human tissues and enterotoxigenic strains of E. coli causing disease in humans. Certain E. coli strains adhere to human mucosal cells by so-called type 1 pili which can be inhibited by the sugar mannose (23). In vitro binding of enteropathogenic E. coli strains to human fetal small intestine has been reported to occur in the presence of 0.5% mannose-hence not due to type 1 pili-and appears to be host specific (21). Evans et al. (8) have shown a close correlation of the presence of hemagglutination of human group A erythrocytes, the presence of the CFA antigen, and colonization of the infant rabbit small bowel. This study was undertaken to screen enterotoxigenic E. coli strains recently isolated from humans with diarrhea for the presence of mannose-resistant hemagglutination (MR-HA) of human group A and B erythrocytes and guinea pig erythrocytes and for adherence to human buccal mucosal epithelial cells. Binding to buccal cells was explored since it has been shown that another enteric pathogen, Vibrio cholerae, is present in high numbers in the oral cavity during acute and convalescent periods (14). Others studying infantile diarrhea due to E. coli have reported a high percentage of positive throat cultures of the E. coli pathogen in children not known to have vomited during their illness (44). Thus, colonization of the oral cavity appears to occur pari passu with upper intestinal colonization in the natural disease caused by these pathogens. MATERLALS AND METHODS
Cultures and cultural conditions. Strains of E. coli used are described in Table 1. The strains from Kenya (the K series) and from Mexico (the TD series) were isolated within 1 and 2 years of study in our laboratory. The Dacca (the D series) strains were sent to us within 1 month of isolation. These strains were kindly supplied by R. B. Sack. For long-term storage, the strains were maintained in liquid form in 20% glycerol at -20'C. Working cultures were kept at room temperature on 2% peptone (Difco) and 0.5% NaCl containing 1.5% agar (PA). Certain of the strains were found to have lost the ability to produce toxins as assessed in the Y1 and suckling mouse assays, but were examined for adherence ability. Such strains are indicated in Table 1 by parentheses around the toxin notation. Bacterial growth for adhesion assay. For human buccal adhesion assays, overnight PA cultures were transferred to fresh PA slants containing 25 ,uCi each of [3H]alanine and [3H]leucine. After growth for 4 to 6 h at 37C, the bacteria were harvested by resuspension in phosphate-buffered saline (PBS), pH 7.2. This suspension was used for the adherence assay. The absorbance at 625 nm of a bacterial sample diluted 1:10 was determined by use of a Perkin Elmer-Cole-

present, using the experimentally determined value 2.5 x 109 colony-forming units/ml gives A625 = 1.0. A sample of the diluted suspension was also filtered through a 0.2-,um Nucleopore filter (Nucleopore Corp., Pleasanton, Calif.) to determine the specific activity of the bacterial cells. Buccal cell preparation. Buccal cells were collected from a constant group of male and female, nonsmoking volunteers by scraping the mucosal surface of the cheeks with wooden tongue depressors. Pools were prepared from 8 to 15 donors to minimize individual variation. The cells were rinsed from the sticks, concentrated by low-speed centrifugation, washed five times, and resuspended in 0.5% mannosePBS. The number of cells per milliliter was determined with a hemacytometer. Buccal cell adhesion assay. The binding of radioactive bacteria to isolated buccal cells was measured by a filtration assay. The bacteria and buccal cells were mixed and incubated for 3 min in a water bath at 37C; duplicate samples were then filtered through prewashed 5.0-,um Nucleopore filters. The unbound bacteria were removed by one washing with 5 ml of 0.5% mannose-PBS. Suction was maintained at less than 1 in. of vacuum by use of an air bleed arrangement. Nonspecific binding of bacteria to the filter membrane was measured by incubating and filtering control samples (without buccal cells). The counts bound to the control filters were subtracted from the counts retained by the filter when buccal cells were present. The filters were counted by use of Aquasol-2 (New England Nuclear, Boston, Mass.) in a Beckman liquid scintillation counter (model LA-3133P). Experimental conditions were internally monitored daily by including strains 334 and 334LL as control adherent and nonadherent strains, respectively. (Strain 334LL is a plasmid-free derivative which is MR-HA negative and does not bind to buccal cells.) Data were discarded when the binding level of strain 334 was not fivefold greater than that of strain 334LL. Analysis of the data was performed by a nonparametric statistical procedure, the Mann-Whitney U test

man model 44 spectrophotometer. The absorbance was employed to calculate the number of bacteria

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(36).
Isolation of pili. Bacteria were grown overnight at 37C on peptone-salt agar (2% Bacto Peptone [Difco], 0.5% NaCl, and 1.5% agar). The cells were harvested in PBS (pH 7.2). The bacterial cells were washed once and resuspended in PBS containing 0.5% mannose. Pili were removed from the washed bacteria by blending for 3 min with the use of short (about 30 s) bursts, the blender being cooled on ice to minimize thermal denaturation. Intact bacteria and large debris were removed by centrifugation at 27,000 x g for 10 min, leaving the pili in the supernatant. This 0.5% mannose-PBS supernatant fluid, containing pili, was then mixed with freshly drawn, washed guinea pig erythrocytes and was cooled on ice to allow attachment of the pili to the blood cells. These red cells were then washed with 0.5% mannose-PBS in the cold, by use of low-speed centrifugation. Thus, only those bacterial components which bound to red cells in the presence of mannose in the cold and sedimented at

692

THORNE, DENEKE, AND GORBACH

TABLE 1. Escherichia coli strains examined
Strain Enterotoxin status'

INFECT. IMMUN.

Serotypeb
015:Hll 015:Hll NT 078:Hll 078:Hll LP

SourceC

Reference

Human strains 334 334LL 193-4 H10407 H10407P TX-1 TX-85 B2C B7A 214-4 H10405 HS CD-1 K324cl K344c2 K135c2 K325c3 K326c5 TD462cl TD213c2 TD235c4 TD219cl TD451c2 TD327c2 M409cl M403c3 D542 D280551 D280561 D370855 D563 D481 D370844 D444 D513 D524 Animal pathogens

LT/ST (LT/ST) LT/ST LT/ST LT(ST) ST ST LT/ST LT/ST ST LT? LT/ST LT (LT) (LT) LT/ST LT/ST ST LT ST LT/ST (LT) ST ST LT/ST LT/ST LT/ST LT/ST LT/ST LT/ST LT/ST LT/ST LT/ST LT/ST
and

AD, India
AD, India AD, India
LP
9-11

078:K80:Hi2 078:K80:Hi2 06:Hi6

0148:H28 NT

ID, Texas ID, Texa' AD, Viet Nam AD, Viet Nam AD, Maryland
Adult feces Adult feces Adult feces

08:060:H9

025:H42

06:Hi6
0128

06:H16
05 NT NT NT NT NT

AD, Kenya AD, Kenya AD, Kenya AD, Kenya AD, Kenya AD, Mexico AD, Mexico AD, Mexico AD, Mexico AD, Mexico AD, Mexico AD, Morocco AD, Morocco AD, Dacca AD, Dacca AD, Dacca AD, Dacca AD, Dacca AD, Dacca AD, Dacca AD, Dacca AD, Dacca
AD, Dacca

11 45 45 5 5 19 11, 18 5 5 32 32 32 32 32 23 23 23 23 23 23

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plasmid-containing
strains RDEC-I Shiga enterotoxin Ovine diarrhea 1 P-307 K88 Porcine diarrhea K-12 LP K-12 K88ab K88ab LD K-12 K99 K99 LD a Parentheses indicate loss of enterotoxin production; -, nonenterotoxigenic strains. b NT, Nontypable. eAD, Adult diarrhea; ID, infant diarrhea; LP, laboratory passage; LD, laboratory derived. low speed were retained. Pili were eluted from the red Pilus antiserum. Antisera were prepared in rabcells under conditions which reversed the MR-HA bits by the procedure of Edwards and Ewing for the reaction, i.e., resuspension in PBS and incubation for preparation of O:K antisera (6). Pilus antiserum for 15 min at 37C. The red cells were removed by cen- strain 334 was prepared as described above, followed trifugation at 500 x g for 10 min in a clinical centrifuge by cross-adsorption with strain 334LL. This allows for at room temperature. This wash-elution procedure removal of antibodies to common 0 and K antigens. was repeated at temperatures of 37, 42, and 50C. Pilus-specific antiserum was also prepared by injection Finally, erythrocyte membrane fragments and debris of the isolated pili suspension described above. were removed by ultracentrifugation in a Beckman Immunoglobulin G from whole rabbit serum was model L5-50 centrifuge at 183,000 x g for 2 h, leaving purified by sequential sodium sulfate precipitation partially purified pili in the supernatant fluid. Chemi- (initially 18%, then 14%) followed by dialysis and pascal characterization and electron microscopic study of sage through diethylaminoethyl-Sephadex in 15 mM these pili are described in a manuscript in preparation. phosphate buffer (pH 8.0) (12). Monovalent Fab frag-

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ment was prepared by overnight papain digestion followed by Sephadex G-100 column chromatography to remove intact immunoglobulin G (40). The purity of the isolated Fab fragment was assessed by the absence of intact heavy chain after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (19). Hemagglutination. Pilus-mediated bacterial hemagglutination of washed guinea pig and human group A and B erythrocytes was tested in the presence of 1% mannose at 40C. Strains were tested after overnight growth at 370C on PA slants (4, 8). Enterotoxin testing: infant mouse assay. E. coli strains were tested for stable enterotoxin (ST) production by use of the infant mouse method of Dean et al. (2). This test has been shown to be specific for ST (16). Culture filtrates from E. coli isolates grown on Casamino Acids-yeast extract medium were given by intragastric injection, directly through the abdominal wall; at least three mice per isolate were used. Positive strains produced intraintestinal fluid accumulation resulting in a ratio of weight of intestine/weight of carcass >0.083. Strains were retested after heating the culture filtrate at 650C for 15 min (13). Adrenal cell assay. All isolates were tested for labile enterotoxin (LT) production by a miniculture modification (33) of the Y1 adrenal tumor cell culture assay described by Donta and Smith (3). Rabbit ileal loop test. Strains found positive in any of the above assays were tested in the adult rabbit ileal loop assay (7). Bacteria were grown in glucose syncase medium at 370C with aeration. A 1.0-ml sample of the sterile culture filtrate was injected into 5-cm ileal loops, and rabbits were sacrificed after 6 or 18 h. Both heated (65C for 15 min) and unheated filtrates were tested in at least three rabbits, and a mean volume/length (milliliters/centimeters) ratio was determined. A positive reaction was scored when the volume/length ratio was -1. Only test results from rabbits with appropriate positive and negative control loops were accepted. Indirect fluorescent-antibody assay. Human buccal cells were prepared as previously described and then were mixed with the appropriate E. coli strain. After 15 min of incubation at 37C, the excess bacteria were removed by differential centrifugation. The mixtures were washed five times with PBS, and the final pellet was resuspended in 50 pl and applied to glass microscope slides. The samples were air-dried and fixed in methanol for 2 min. Slides were stained for 30 min with appropriately diluted (1:10) rabbit antiserum specific for the test organism. The slides were then rinsed and washed in PBS for 1 h to remove excess rabbit serum. The fluorescent probe was goat-antirabbit serum conjugated with fluorescein isothiocyanate (FITC; Gibco, Grand Island, N. Y.). This conjugate was diluted (1:50 to 1:100) and applied for 30 min. The slides were again rinsed, washed, and mounted in glycerol-PBS (2:1). Care was taken to prevent exposure of the FITC-labeled material to light, which leads to photodecomposition. A Nikon fluorescence microscope equipped with an FITC interference filter (495 nm) was used to examine the slides. An interference filter was necessary for unequivocally distinguishing scattered from emitted light.

Agarose gel electrophoresis. Deoxyribonucleic acid was isolated from the strains by use of the crude lysate method described by Meyers et al. (22). Electrophoresis of the ethanol-precipitated deoxyribonucleic acid was performed in 0.7% agarose dissolved in tris(hydroxymethyl)aminomethane-borate buffer, pH 8.25. A dye solution was added to the deoxyribonucleic acid samples prior to electrophoresis. The dimensions of the gel were 9.6 by 14.2 by 0.15 cm; 25-Ad samples were run. The power source was a Shannon regulated high-voltage power supply. Electrophoresis was carried out at 60 mA, 120 V for 1.5 to 2 h. The gel was stained with ethidium bromide (0.4 ,ig/ml) in tris(hydroxymethyl)aminomethane-borate buffer and photographed with the use of a red-orange filter.

RESULTS MR-HA and pilus-agglutination reactions of enterotoxigenic E. coli strains. The strains described in Table 1 were examined for their ability to cause hemagglutination of human group A and B and guinea pig erythrocytes in the presence of 1% mannose at 4C (MR-HA). These conditions are believed to allow for detection of pili that assist in bacterial colonization of the small bowel. As shown in Table 2, two strains (334 and 193-4) caused MR-HA of human A and B and guinea pig erythrocytes. Twelve strains gave strong MR-HA reactions with both human A and B erythrocytes, but not with guinea pig cells. Three test strains were MR-HA reactive only with guinea pig erythrocytes. Agglutination of these strains by antiserum directed against 334 pili was also examined. Strain 334 (015:H11), isolated in Calcutta from a patient with severe diarrheal disease, produces both ST and LT enterotoxins. Analysis of a sodium dodecyl sulfate crude lysate by use of an agarose electrophoretic technique demonstrated the presence of six plasmid bands. A plasmidfree derivative of 334 called 334LL (015:H11) is nontoxigenic and MR-HA negative. This strain was used to cross-absorb 334 antigens. This absorbed antiserum was found to cause the agglutination of 11 other strains in our collection (Table 2). Of the 12 strains that reacted with the specific antisera, 11 were also MR-HA' with either human or guinea pig red blood cells; the one exception was H10407P. Seven strains were MR-HA' but unreactive in the antiserum. All strains reacted identically in the more specific antisera raised against the surface structures (pili) present on strain 334. The strains agglutinated by 334 pili antisera belonged to several 0 serotypes, 078:K80:H12, 078:H11, 025:H42, which are not cross-reactive (28). The 078 strains, however, have the same flagellar antigen, H11, as strain 334. Buccal cell adherence assay. Buccal cell adherence was determined by use of a radioac-

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INFECT. IMMUN.

TABLE 2. Comparison of agglutination reaction and/or buccal cell adherence of human toxigenic E. coli strains
Strain

Serotypea
015:Hii NT 078:K80:H12

MRHAb B

Buccal adherence GP

334i

an-

tiseruM
+ +

Group I 334
193-4

+ +
+

+ +
+

TX-85 TX-1 K324cl H10407 K326c5 D542 D563 D481 D280551 Group II H10407P K325c3 K135c2 TD235c4 TD462cl 214-4 B2C D444 Group III TD213c2 TD219cl D370855 D370844 TD327c2 D280561 Group IV B7A M403c3 K344c2 TD451c2 D513 D524 M409cl Control, nontoxigenic 334LL CD-1 HS RDEC-1 P307 K-12 K-12 K88 K-12 K99 a NT, Nontypable.

078:K80:Hi2 08:060:H9 078:Hi1 025:H42 NT

NT NT NT 078:Hii

+ + + + + + +

+ + + + + + +

+ + +

+ +
_ -

246 171 + i01 147 172+ 253 136 115 102 135 128

48 30 10 28 66 73 9 11 37 37 20

(11) (3) (4) (4) (3) (4)

+ + + + + + + + +
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(2)
(3) (3) (3) (4) (7)

06:H16 NT 06:H16 NT
0128 06:H16 NT NT

_ -

_ + + + + _ -

255 104 132 55 470 56 144 39 275 86 108 14 183 40 112+ 21

(4)
(3)

(5)
(3)

(5) (4) (4)

05 0148:H28

+ + + + _ _ -

46 6 (7) 50 28 (3) 50 10 (6) 39 5 (4) 30 20 (2) 86 14 (4)

75 28 84 76 22 17 25

58 11 42 15 15 9 5 3 9 17 14 29 15 38 19

(4)
(7) (5)

(4)
(4) (3)

(4)
(10) (5) (5)

015:H11

+ + -

13 37 61 43 38 31 81 48

(4)
(3) (4)

(4) (4)

bMannose-resistant hemagglutination (MR-HA) of washed human group A (A), group B (B), or guinea pig (GP) erythrocytes at 0C. 'Counts of number of bacteria/buccal cell standard error of the mean. The number of assays is shown in parentheses. d Agglutination by rabbit antisera prepared against surface structure present on strain 334.
quently, at least two control strains, one binder and one nonbinder, were included in each series of tests (Table 2). The adherence reaction was
rapid, and all test strains were incubated for 3

tive filtration assay. Strains were repeatedly tested with buccal cells pooled from a minimum of eight donors. Day-to-day variations were observed in the level of bacterial binding. Conse-

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min with the buccal cell preparation. Of the 32 toxigenic test strains, 19 were found to adhere at a level similar to that of strain 334. The mean net number of attached cells per buccal cell of adherent strains ranged from 101 to 470. Toxigenic nonbinders averaged from 17 to 86 attached bacterial cells per buccal cell. The nontoxigenic human strains tested did not adhere to any significant degree. Strain 334LL, the plasmid-free MR-HA- derivative of 334, showed very low binding. The animal pathogens P307 and RDEC-1 and the E. coli K-12 strains containing the K88 and K99 plasmids did not adhere to any significant degree. Comparison of the binding of human isolates of toxigenic E. coli strains (groups I-IV) to the control organisms, by the Mann-Whitney U test, revealed that the toxigenic strains had a significantly higher level of adherence to human buccal cells (P = 0.007). Fab specific for 334 pili was found to block binding of strain 334 to buccal cells (Table

3).

Originally, strain H10405 was included as a control nontoxigenic strain and was found to be buccal adherent (259 93 bacteria/buccal cell, four assays). H10405 was neither red cell reactive nor agglutinated by 334 pili antisera. Recently Klipstein et al. (18) reported a low level of toxin production by this strain. Strain H10405 was

originally isolated from a case of undiagnosed adult diarrhea and contains plasmids. Consequently, its use as a control strain is questionable. Fluorescent staining of adherent bacteria. A fluorescent-antibody technique was also used to evaluate bacterial binding to individual buccal cells. With the strains tested, there was good agreement between the fluorescent-stained preparations and the radioactive filter assay. Strains 334 and 193-4 were seen to coat the buccal cells heavily (Fig. 1). The negative control strains K-12, K-12 K99+, HS, and 334LL did not appear to bind, as shown in Fig. 2. The K-12 K88+ strain did adhere in small numbers but was never seen to coat the buccal cell surface as did human enterotoxigenic strains tested. Correlation of MR-HA, buccal cell adherence, and presence of 334 surface antigen. There was no direct correlation between adherence to human buccal cells and MR-HA of any one of the three blood types used (i.e., human A or B and guinea pig). Therefore, strains are described as being MR-HA+ if they react with any of the erythrocytes employed. The strains examined in this study can be placed into four groups based on ability to hemagglutinate erythrocytes of humans or guinea pigs and/or to bind to human buccal cells (Table

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FIG. 1. Fluorescein-labeled E. coli 193-4 and human buccal cells. x200.

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FIG. 2. Fluorescein-labeled E. coli K-12 and human buccal cells. x400.

TABLE 3. Inhibition of buccal binding of strain 334 by anti-334 MR-HA pili Fab Mean net no. of bacteria/bucF~b (.,I~a cal cell

DISCUSSION

F,,b (jilYa
_ 200 400 200*

100 60 10 110 a Bacteria alone were pretreated for 15 min with Fb preparation or (*) heat-inactivated normal rabbit serum.

2). The first group is composed of strains which were MR-HA' and adhered to buccal cells; 10 of the 11 strains also reacted with 334 pili antisera. The second group are strains which were MRHA- and adhered to buccal cells but, with the exception of strain H10407P, were unreactive in the antisera. The third group are strains which were MR-HA' but did not adhere to the human buccal epithelial cells. The fourth group are toxigenic E. coli strains which were MR-HAand did not adhere to the human buccal cells.

In our study of enterotoxigenic E. coli strains isolated from patients in various parts of the world, we encountered strains of several serotypes which varied in their ability to cause hemagglutination of human and/or guinea pig erythrocytes, adhere to human buccal cells, and be agglutinated by specific 334 pili antisera. One group of strains possessed all of these characteristics. A second group was able to adhere to buccal cells but did not cause MR-HA. There was a third group of six MR-HA positive strains which did not bind to the buccal cells. These data suggest that multiple surface "sticky" antigens (adherence factors) are present on strains of toxigenic E. coli isolated from humans. Binding to human buccal cells, in the presence of mannose, appears specific for strains isolated from humans. The E. coli animal pathogens P307 (a porcine pathogen) and RDEC-1 (a cause of diarrhea in rabbits) and the plasmid-containing strains K-12 K99 and K-12 K88 show only background level adherence in the buccal assay.

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All MR-HA' strains examined were found to react strongly with human type A and B erythrocytes and/or guinea pig erythrocytes; the latter property was used previously to detect the K88 antigen (41, 42). Use of guinea pig blood allowed for the detection of strain K324cl, which reacts only with this erythrocyte type and yet adheres strongly to human buccal cells. Although some adherent strains can also cause MR-HA of various erythrocytes, the occurrence of MR-HA negative buccal binding E. coli strains cautions against complete reliance on the MR-HA phenotype to detect E. coli strains which adhere to the human mucosal tissues. It should be emphasized that the hemagglutination and buccal adherence assays were performed in the presence of D-mannose, a sugar known to inhibit binding by type 1 E. coli pili (25, 34, 35). Thus, the mannose-resistant interaction between these organisms and the surfaces of human buccal and red blood cell membranes does not involve type 1 pili, but must be some other surface components which may or may not be pili. Additional studies indicate that organisms in group I possess surface structures which appear pilus-like by electron microscopy (manuscript in preparation). These specific pili appear to adhere to eucaryotic membranes in the presence of mannose; adherence is blocked by pili-specific, cleaved immunoglobulin G antibody. In their extensive study of E. coli strain H10407 (078:H11), Evans and co-workers (8-11) have documented the production of a fimbriate structure, called colonization factor antigen (CFA). The CFA allows for MR-HA of human erythrocytes (particularly blood group A) and promotes colonization of the upper small bowel of the infant rabbit. Recently, the CFA-positive strain H10407 was shown to cause diarrheal disease when fed to human volunteers, in contrast to the CFA-negative strain H10407P, a spontaneous derivative of H10407. Although these two strains differ in their MR-HA ability, we have found that both are able to bind to buccal cells in the presence of mannose and both agglutinate with our 334 pili antisera. No explanation can be offered at present. We plan to examine strain H10407P for the presence of other adherence antigens, unrelated to CFA. Recently, 0rskov and 0rskov (27) studied O:K:H serotypes of 64 enterotoxigenic E. coli strains for the occurrence of CFA. CFA was demonstrated in 078:H, 078:H11, and 078:H12 strains, but not in any strains of the other common serotypes isolated from cases of human diarrhea. MR-HA ability of these strains was also tested, as in our study some strains were

found which could only cause MR-HA of guinea pig or human red blood cells. As noted in Table 2, we found four strains belonging to different serotypes which cross-reacted with 334 pili antiserum. Other toxigenic E. coli strains previously used in human challenge experiments were included in this study. Strain 214-4, which was shown by Levine et al. (20) to cause disease in adults, and strain B2C, which was similarly described by DuPont et al. (5), were both found to bind to human buccal epithelial cells. Another test strain, B7A, did not appear to bind. Spontaneous loss of toxin production, hemagglutinating ability, and CFA are well documented and could account for such variations (11, 14, 26). During the course of our studies, we have encountered similar events, i.e., simultaneous loss of MR-HA ability, buccal binding activity, and agglutination by 334 pili specific antisera; analysis of these strains revealed a concurrent loss of multiple plasmids. A study of the role of plasmids in mediating MR-HA ability and buccal binding is in progress. Little is known about receptors on the buccal cells in this assay. Direct visualization of the adherent bacteria, by use of a fluorescent-antibody technique, has revealed some variation in binding among cells of the buccal "pool." Strain 334 was seen to coat the entire surface of certain buccal cells in the "pool," whereas strain 193-4 adhered in large numbers on certain areas of buccal cells. Variations in receptors on buccal mucosal cells have been noted in other binding studies (24). Although the in vitro buccal adherence system cannot duplicate in vivo growth and competitive colonization of the small bowel, the assay does allow for studies of the early events in bacterial adherence to mucosal cells of human origin. The reaction of group I strains with erythrocytes raises the possibility that these organisms are adhering to ABO and Lewis blood group receptors or similar structures. These substances are known to be present in secretions and on other tissues, including those of the entire intestinal tract (31, 43). Since buccal mucosal cells share certain similarities with the gastrointestinal mucosa, this model provides a system for increasing our understanding of the complex interaction between bacterial attachment components and receptors on eucaryotic cell membranes.
ACKNOWLEDGMENTS
This investigation was supported in part by Public Health Service research grant R22AI13164-02 from the National Institute of Allergy and Infectious Diseases and by contract 22375-2116 from the Food and Drug Administration and contract DAMA 17-76-C-6007 from the Department of the United

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