Vol. 24, No. 4JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1986, p. 512-5160095-1137/86/100512-05$02.00/0Copyright © 1986, American Society for Microbiology

Enzyme-Linked Immunosorbent Assay for Adherence of Bacteriato Animal Cells

ITZHAK OFEK,1 HARRY S. COURTNEY23* DIETER M. SCHIFFERLI,2 AND EDWIN H. BEACHEY2'3 4

Department of Human Microbiology, Sackler School of Medicine, Tel Aviv, Israel'; Departments of Microbiology andImmunology2 and Medicine,4 University of Tennessee, Memphis, Tennessee 38103; and Veterans Administration

Medical Center, Memphis, Tennessee 381043*

Received 14 April 1986/Accepted 13 June 1986

Epithelial cells scraped from human oral mucosa and from pig intestines were immobilized onto the flatbottom surfaces of microtiter plates to study the adherence of various bacterial species to host cells. Bacterialadherence was quantitated either by an enzyme-linked immunosorbent assay technique with specific antibac-terial serum as the first antibody followed by peroxidase-conjugated second antibody or by using biotinylatedbacteria and avidin-peroxidase as the detecting agent. Unlabeled Escherichma coli and purified E. coli 987Pfimbriae inhibited the adherence of biotinylated E. coli to immobilized enterocytes. The adherence of a

mannose-sensitive strain of E. coli to immobilized oral epithelial cells was inhibited by mannose derivatives.The adherence of fimbriated E. coli 987P to immobilized enterocytes was approximately four times higher thanthe adherence of a nonfimbriated variant of the same strain. The adherence of Streptococcus pyogenes to oralcells was detected in the range of 10 to 150 bacteria per cell and was inhibited by lipoteichoic acid and albumin.The data suggest that the putative receptors which bind bacteria on the immobilized cells retain a functionalform similar to that of native cells in suspension. The proposed adherence assay is easy to perform, allows thedetection of specific adherence of test bacteria, and provides objective quantitation of adherence with a

sensitivity of 10 bacteria per cell. Most importantly, the assay allows the testing of many variables in the sameday.

In the last decade, various methods have been devised tostudy the adherence of bacteria to various nonphagocyticcells. Three types of animal cells have been used: cells fromintact excised tissue (20), tissue culture cells (13, 18), andcells in suspension obtained from scraped tissues or fromexfoliated cells (16) as initially described by Duguid andGillies (7). The binding of bacteria to these cells has beenquantitated by direct microscopic counting of adherent bac-teria, counting of radiolabeled bacteria, and enumeration ofCFU associated with the cells. Adherence assays are per-formed by mixing bacterial suspensions with the animal cellsand then separating the cells with adherent bacteria from thenonadherent bacteria. Various techniques have been usedfor separation, such as differential centrifugation, densitygradient centrifugation, and filtration through 8-[Lm-pore-size filters or, in the case of tissue culture cells and intactexcised tissues, by sequential washing.Two major issues must be considered in selecting the

method to be used: the availability of the test animal cellsand the reliability of the technique used to separate cells withattached bacteria from the nonadherent bacteria. The tissueculture method can achieve adequate separation but requirestissue culture facilities that frequently are not available inbacteriological laboratories, and the cell types available arenot always germane to the particular interest of the investi-gator. The excised tissue method can also achieve adequateseparation but has limited sources and has been confinedprimarily to the intestinal tract. In contrast, scraping cellsfrom tissues or collecting exfoliated cells can provide manycells from a wide variety of tissues. The separation of animalcells with adherent bacteria from nonadherent bacteria byeither differential centrifugation or filtration through >8-[Lm-pore-size filters has been difficult to achieve. In addition,

* Corresponding author.

reproducible, quantitative recovery of the animal cells con-taining adherent bacteria is virtually impossible. Most inves-tigators, therefore, have used the tedious and somewhatsubjective microscopic counting of the average number ofadherent bacteria per animal cell or the percentage of animalcells containing a minimum number of adherent bacteria.Although each of the above methods has been useful inobtaining information on the mechanisms of bacterial adher-ence to animal cells (17), none of them is practical for thequantitation of bacterial adherence on a large scale. Forexample, to test the effect of five concentrations of each offive different potential inhibitors on the adherence of twobacterial strains to a desired tissue cell requires 50 testsamples.

In this study, we developed a method by which cells fromvarious mucosal tissues were immobilized on the bottoms ofmicrotiter plates to quantitate bacterial adherence. After thecells were exposed to bacteria, the rnonadherent bacteriawere washed off and an enzyme-linked immunosorbentassay (ELISA) with specific antibacterial serum was used todetermine the total number of bacteria adherent to theimmobilized cells. Alternatively, adherence was estimatedby using biotinylated bacteria. The assays were reproducibleand easy to perform and, most importantly, allowed thehandling of many test samples on the same day.

MATERIALS AND METHODSReagents. Unless specified otherwise, all reagents and

chemicals were purchased from Sigma Chemical Co., St.Louis, Mo. Peroxidase-conjugated goat anti-rabbit immuno-globulin G was obtained from Cooper Biomedical Inc., WestChester, Pa. Lipoteichoic acid was prepared from Strepto-coccIus pyogenes 1RP41 by extraction with phenol andpurification on a Sepharose 6B column as described previ-ously (4).

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Bacterial strains. S. pyogenes M type 5 was grown inTodd-Hewitt broth (Difco Laboratories, Detroit, Mich.) for16 h at 37°C. The bacteria were harvested by centrifugationand washed in phosphate-buffered saline (PBS; 0.15 MNaCl, 0.02 M P04 [pH 7.2]). Stock solutions of S. pyogeneswere stored at -20°C and were thawed and washed oncewith PBS before use in the adherence assays. A urinary tractisolate of a type 1 fimbriated, mannose-sensitive strain ofEscherichia coli M (16) was grown in brain heart infusionbroth (Difco) for 24 h at 37°C, washed in PBS, and sus-

pended to the desired concentration. A diarrheagenic E. colistrain, 987P (09:K103:H-) was a gift from R. E. Isaacson(15). Fimbriated and nonfimbriated variants of E. coli 987Pwere selected from fresh blood agar plates as described byNagy et al. (15). The bacteria were grown overnight intryptic soy broth (Difco) at 37°C, washed in PBS, andsuspended to the indicated concentration.

Purification of E. coli 987P fimbriae. Fimbriae were puri-fied from E. coli 987P by a modification of the technique ofKorhonen et al. (12). The E. coli cells were grown in trypticsoy broth overnight at 37°C with agitation, harvested bycentrifugation, and washed in 10 mM Tris hydrochloride (pH7.8). The E. coli cells were subjected to maximum speed ina blender (Oster, Milwaukee, Wis.) for 2 min. This was

repeated five times with intermittent cooling periods. Theparticulate fraction was removed by centrifugation, and thefimbriae were precipitated with 12% ammonium sulfate. Theprecipitate was dialyzed against 10 mM Tris hydrochloride(pH 7.8)-0.5% deoxycholate and centrifuged on a 10 to 60%sucrose gradient for 20 h at 22,000 rpm on an SW28 rotor(64,000 x g). The fractions containing fimbriae were pooledand dialyzed against 10 mM Tris hydrochloride. Deoxycho-late was removed by the batch technique with AmberliteXAD-2 beads (Polysciences Inc., Warrington, Pa.). Thefimbriae were judged to be pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by electron micros-copy.

Antisera. Rabbit antisera against pepsin-extracted M5protein from group A streptococci and against E. coli wereprepared as previously described (5, 16). 09 antiserum was

purchased from Difco.Biotinylation of E. coli. E. coli 987P was grown as de-

scribed above, washed in PBS, and suspended in 0.1 Mcarbonate (pH 8.2) to an optical density at 550 nm (OD550) of0.7. A sample (12.5 ,u1) of biotin-n-hydroxysuccinimide (10mg/ml in dimethyl sulfoxide) was added to 1 ml of thebacterial suspension and incubated for 2 h at ambient tem-perature. The E. coli cells were then washed three times withPBS and suspended in PBS to the indicated concentration.

Preparation of epithelial cells. Oral epithelial cells (OEC)were collected from human donors by swabbing the buccalepithelium with cotton swabs. The swabs were vigorouslystirred in PBS to release the epithelial cells. The epithelialcells were pooled, washed, and resuspended in PBS to aconcentration of 1.5 x 105/ml. Enterocytes were isolated byscraping the pig small intestines by the method of Dean andIsaacson (6). The cells were suspended in PBS at a concen-tration of 2 x 106/ml.

Immobilization of cells onto microtiter plates. To each well(96-well, flat-bottomed microtiter plates; Linbro/Titertek;Flow Laboratories, McLean, Va.), 100 of a 1 M lysinesolution in distilled water was added and incubated for atleast 10 min and up to 2 h at room temperature. The plateswere then washed three times with distilled water, and 100of a 1.25% glutaraldehyde solution in distilled water wasimmediately added. The plates were incubated for exactly 5

TABLE 1. Blocking by various agents of nonspecific binding ofS. pyogenes to activated plates

Blocking agenta Concn % Inhibition of binding ± SDb

Gelatin 2 mg/ml 99 ± 0.5Amino acid mixturec 1 mg/ml 10 ± 5.6Tween 20 0.01% 95 ± 2.1Horse serum 10% 39 ± 5.2BSA 50 mg/ml 84 ± 1.5Hemoglobin 20 mg/ml 94 ± 1.9Casein 20 mg/ml 43 ± 4.8

a All agents were dissolved in PBS.b The ELISA values for binding to activated plastic in the absence of

blocker ranged from 1.77 to 2.30. Percentage of inhibition = [1 - (values ofblocked wells/values of unblocked wells)] x 100.

c A mixture of 20 amino acids, each at 1 mg/ml.

min and then were washed two times with distilled water.Microtiter plates at this stage were termed activated. PBS(100 p,l) was immediately added to each well, followed by 50,ul of the epithelial cell suspension. The cells were gentlymixed and allowed to settle for 10 min at ambient tempera-ture. The plates were then centrifuged for 10 min at 650 x g.The supernatant was aspirated with caution so as not todisrupt the cell monolayer. The plates were then driedovernight at 37°C and stored at room temperature until used.Control plates were prepared as described above but withoutepithelial cells.

Assay of blocking agents. Various agents were tested fortheir ability to block nonspecific binding of S. pyogenes toactivated plastic. Various agents (Table 1) were dissolved inPBS at the indicated concentrations, and 100 RI1 of each wasadded to the wells of activated plates. The plates wereincubated at 37°C for 1 h and washed five times with PBS,followed by the addition of 100 ,ul of the streptococcalsuspension and adjustment to an OD530 of 0.4 in PBS. Theplates were rotated horizontally for 1 h at ambient temper-ature, washed five times with PBS, and heat-fixed for 10 minat 65°C. The bound bacteria were enumerated by the ELISAtechnique as described below.Adherence assay with biotinylated bacteria. The unoccu-

pied plastic space in wells containing immobilizedenterocytes was blocked by adding 100 ,ul of a 5% solution ofbovine serum albumin (BSA). The plates were incubated for3 h at 37°C and washed three times in PBS. A sample (50 ,u1)of biotinylated E. coli (approximately 2 x 108 ml) was addedto appropriate wells and incubated for 40 min at 37°C. Theplates were washed three times with PBS and heat-fixed for10 min at 65°C. Avidin-peroxidase conjugate (100 pl; 200ng/mI in 5% BSA) was added to each well and incubated for30 min at 37°C. The plates were then washed three timeswith PBS, and 100 pL. of O-phenylenediamine solution wasadded to each well. The color was allowed to develop for 15to 30 min, and the A450 was measured with a microtiter platereader (Dynatech Laboratories, Inc., Alexandria, Va.). Thecontrols consisted of wells treated as described above butwithout immobilized enterocytes or without biotinylatedbacteria to determine nonspecific binding. To determinewhether biotinylation of the bacteria affected the adhesiveproperties of the E. coli cells, serial twofold dilutions ofunlabeled bacteria were added to each well before theaddition of the biotinylated bacteria. The specificity of thebinding of biotinylated bacteria to the immobilizedenterocytes was determined by mixing increasing concentra-tions of purified fimbriae with the biotinylated E. coli cells.Adherence assay with antibodies. Before the adherence

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FIG. 1. Correlation between ELISA values for adherence andthe number of bacteria attached to epithelial cells. Increasingnumbers of S. pyogenes cells were added to the wells of a microtiterplate as described in Materials and Methods. The adherent bacteriawere enumerated by an ELISA (O) or by direct microscopic count

(O).

assay, all wells were incubated with 100 RI of blocking agentfor 1 h at 37°C. The wells were then washed four times withPBS. The bacteria were adjusted to the indicated concentra-tion in PBS, and 100 ,ul of the solution was added to eachwell. The microtiter plates were rotated horizontally for 1 hat ambient temperature and then washed five times withPBS. After the last wash was removed, the plates were fixedby heating at 65°C for 10 min. A sample (100 ,ul) of theindicated dilution of rabbit antibacterial serum in 5% BSAwas added to each well, incubated for 1 h at 37°C, andwashed five times with PBS. Peroxidase-labeled goat anti-rabbit immunoglobulin G (100 pA) diluted 1/1,000 in PBS-5%BSA was then added to each well and incubated for 1 h at37°C. After five washes with PBS, 100 pul of a solution of5-aminosalicylic acid was added to each well. The color wasallowed to develop for 15 to 30 min and was then measuredat 450 nm with a microtiter plate reader (Dynatech). Controlsconsisted of wells without bacteria to ensure that the pri-mary antibody did not cross-react with the endogenousbacteria or with the host cells and wells without immobilizedcells to determine the amount of nonspecific binding of thebacteria to plastic.

RESULTS

The number of cells immobilized on the flat bottoms ofmicrotiter plates was approximately 7 x 103 to 8 x 103 forOEC and approximately 13 x 103 to 14 x 103 forenterocytes. The OEC were approximately twice the size ofenterocytes, and this seemed to affect the cell density, whichwas calculated to be 60 to 70/mm2 and 120 to 140/mm2 forOEC and enterocytes, respectively. Cell density was an

important factor in the assay because of the possible non-

specific adherence of bacteria to unoccupied plastic space.

We estimated that in the case of OEC about half of theplastic surface was occupied by the immobilized cells.Therefore, the ELISA for wells without epithelial cellsoverestimated by approximately 50% the nonspecific bindingof bacteria to plastic. Thus, any difference in ELISA values

between wells containing immobilized cells and those with-out cells is a reflection of specific adherence to host cells.The choice of a blocking agent is crucial. We tested

various agents for the ability to block nonspecific binding ofbacteria to activated plastic (Table 1). Gelatin, BSA, Tween20, and hemoglobin were the best blocking agents tested.The choice of blocking agent for adherence assays wasguided by the ability of the agent to block more than 80% ofnonspecific binding to plastic and by consideration of anyeffects that the agent may have on specific adherence to theimmobilized cells.We used hemoglobin for streptococcal adherence assays

and BSA for assays of gram-negative bacteria. By usingthese two proteins as blockers for nonspecific adherence ofbacteria to plastic, we obtained low background ELISAreadings in wells without epithelial cells. These backgroundreadings were subtracted from the higher values to obtain aquantitative measure of bacterial adherence to epithelialcells. Such an approach probably overestimates the valuesfor nonspecific adherence of bacteria to plastic in wellscontaining immobilized cells, especially in wells containing aconfluent monolayer of cells, but it seems to be the mostpractical approach to rule out any possible effect of nonspe-cific adherence of bacteria to plastic.The adherence of S. pyogenes to immobilized OEC was

dose dependent from 106 to 109 bacteria per ml (Fig. 1). Todetermine whether the ELISA readings corresponded tobacterial adherence to cells, the bottoms of the wells wereremoved and the number of adherent bacteria per epithelialcell was counted microscopically. There was a good corre-lation between the ELISA readings for adherence and thoseobtained by direct microscopic counts, and adherence as lowas 10 bacteria per cell was detected by the ELISA technique(Fig. 1). This suggests that the ELISA is reliable for mea-suring the adherence of bacteria to animal cells.To determine whether the mechanisms of bacterial adher-

ence to immobilized cells are the same as those involved inthe adherence of bacteria to cells in suspension, we testedthe inhibitory activity of various agents that were previouslydemonstrated to specifically block the adherence of bacteriato cells in suspension. Methyl-a-D-mannoside and p-nitrophenyl-mannoside were used as specific inhibitors forthe mannose-sensitive adherence of E. coli (8), and BSA andlipoteichoic acid were used as inhibitors of streptococcaladherence (3, 17). These inhibitors blocked the adherence ofthe corresponding bacteria, suggesting that the molecularmechanisms of adherence of E. coli and S. pyogenes toimmobilized cells and cells in suspension are similar (Table2).

Cells other than OEC may be immobilized onto microtiterplates. We immobilized enterocytes from the intestinalmucosa of pigs and assayed the adherence of E. coli 987P.The immobilized enterocytes bound significantly more of thefimbriated E. coli 987P than of the nonfimbriated phenotype(Table 2). Similar results were obtained previously by usingcells in suspension (10). In addition, biotinylated E. coli wasused to study bacterial adherence to enterocytes. Biotinyl-ated E. coli bound to immobilized enterocytes in a dose-dependent manner, and the binding was inhibited by unla-beled bacteria (Fig. 2), suggesting that the biotinylation ofthe bacteria did not alter the binding properties of the E. coli.Purified 987P fimbriae inhibited the attachment of thebiotinylated E. coli to immobilized enterocytes in a concen-tration-dependent manner (Fig. 3). Isaacson et al. (10) ob-tained similar results with native E. coli and enterocytes insuspension. These results suggest that biotinylation may be

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TABLE 2. Inhibition of adherence of different bacterial strains to cells from various tissues immobilized onto microtiter plates

Bacterium Cell type Inhibitor Concn Adherence

S. pyogenesb OEC 0.354OEC Lipoteichoic acid 1 0.061OEC BSA 1 0.055

E. coli Mc OEC 0.389OEC Methyl-a-D-glucoside 25 0.316OEC p-Nitrophenyl-ca-D-mannoside 0.5 0.056OEC Methyl-a-D-mannoside 25 0.041

E. coli 987p+d Enterocytes 0.817E. coli 987p-d Enterocytes 0.240

a ELISA units, A450 of the reduced substrate.SS. pyogenes cells were used at an OD,40 of 0.4, and anti-pepsin-extracted M5 protein serum was used at a 1:400 dilution. Hemoglobin (20 mg/ml) was the

blocking agent.c E. coli with mannose-sensitive type 1 pili was determined by yeast cell agglutination. The E. coli M was used at an OD540 of 0.4, and anti-E. coli M serum was

used at a 1:400 dilution. BSA (50 mg/ml) was the blocking agent.d Fimbriation (P+) or lack of fimbriation (P-) was determined by electron microscopic examination of negatively stained samples. E. coli 987P was used at an

OD540 of 0.5. BSA (50 mg/ml) was the blocking agent. Antisera to E. coli 09 were used at a dilution of 1:1,000.

used in lieu of bacterium-specific antibodies and that immo-bilization of enterocytes on microtiter plates does not alterthe functional properties of their receptor.

DISCUSSIONNumerous studies (for reviews, see references 1, 2, 8, 11,

and 17) have confirmed that bacterial adherence to targettissue cells is an important initial step in the infectiousprocess and involves a stereospecific interaction betweenbacterial adhesins and complementary receptor moleculeson the animal cell surface. The identification of an adhesinand its receptor have allowed the design of in vivo experi-ments aimed at preventing infection. For example, experi-mental infections in animals have been prevented by usingsoluble analogs of the receptor or the adhesin that compet-itively inhibited adherence or by using antibodies raisedagainst the bacterial adhesin or the host cell receptor (1, 2,8). Usually, the identity of a receptor or adhesin is deducedfrom information obtained from experiments in which poten-tial inhibitors or chemical and physical treatments of cellsare tested for their effects on bacterial adherence. Suchtesting would be facilitated by an adherence assay that is

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FIG. 2. Inhibition of adherence of biotinylated E. coli toenterocytes by unlabeled E. coli. Serial twofold dilutions of unla-beled E. coli 987P were added to biotinylated E. coli 987P (approx-imately 2 x 108/ml), and the mixtures were added to the wells of amicrotiter plate containing immobilized enterocytes. The adherenceassay was performed as described in Materials and Methods. Thepercentage of inhibition was calculated by the formula [1-(A450) ofthe test well/A450 of the control well)] x 100.

easily performed, provides objective results, and is readilyavailable to most research and clinical laboratories.The method described in this study for assaying bacterial

adherence to animal cells has some advantages which makeit suitable as the assay of choice in many laboratories. First,and probably most important, many variables may be testedon the same day, i.e., type of host target cell, type ofbacterial strain, and various treatments or potential inhibi-tors. Second, the adherence is evaluated by the objectivecriteria of an ELISA, which avoids the use of radioactivityyet can achieve similar sensitivity. Third, the sources of theimmobilized cells can be diverse, and the immobilized cellscan be kept for relatively long periods. Immobilizedenterocytes or OEC that were stored for 3 to 4 weeksexhibited adherence properties similar to those of cellsimmobilized on day 1 (data not shown). The method can beused to study not only bacterial adherence but also thebinding of bacterial products to tissue cells. In preliminaryexperiments, we found that the B fragment of the E. coli

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987P fimbriae, ,ug/mlFIG. 3. Inhibition of adherence of biotinylated E. coli by purified

987P fimbriae. Increasing concentrations of purified 987P fimbriaewere added to the wells of a microtiter plate containing immobilizedenterocytes. Biotinylated E. coli 987P (approximately 2 x 108/ml)was added, and the adherence assay was performed as described inMaterials and Methods. The percentage of inhibition was calculatedby the formula [1-(A450 of the test well/A450 of the control well)] x100.

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labile toxin was able to bind to immobilized enterocytes(unpublished observation).Other assays for the rapid detection of bacterial adhesins

have been described. Hemagglutination of bacteria is asimple and convenient method that can be used in mostlaboratories to monitor bacteria for adhesive activities andallows the screening of many potential inhibitors (14). How-ever, the test may lead to erroneous conclusions, becausemany strains of bacteria cause hemagglutination yet adherepoorly to epithelial cells (9, 11, 19). Furthermore, bacteriamay produce two adhesins, only one of which is involved inhemagglutination reactions (9, 11, 19). Clearly, there is aneed for a convenient and reliable assay for adherence whichuses desired host cells to screen the adherence of bacterialpathogens, especially when the bacterial receptor is confinedto a particular tissue.

In this study we used three different bacterial strains, eachof which binds to a different receptor. Our results withspecific inhibitors or adherent and nonadherent variants ofthe same strain were compatible with results obtained by thestandard assay with cells in suspension. These results sug-gest that the receptors on the immobilized cells retain theirfunctional form. As with other in vitro assays, includinghemagglutination, the functional activity of these putativereceptors must be confirmed in vivo. However, as a rule,such confirmation is not done until some information aboutthe receptor and the corresponding adhesin is obtained by invitro techniques.

In summary, the proposed assay employing immobilizedanimal cells on the flat bottoms of microtiter plates andELISA techniques allows for reproducible quantitation forscreening as well as for characterizing the molecular mech-anism of bacterial adherence of many bacterial pathogens todiverse types of tissue cells.

ACKNOWLEDGMENTS

This study was supported by research funds of the VeteransAdministration and by Public Health Service grants AI-13550,AI-07238, and AI-10085 from the National Institutes of Health.

LITERATURE CITED1. Beachey, E. H. 1981. Bacterial adherence: adhesin receptor

interactions mediating the attachment of bacteria to mucosalsurfaces. J. Infect. Dis. 143:325-345.

2. Beachey, E. H., B. Eisenstein, and I. Ofek. 1982. Adherence andinfectious diseases. Current concepts. The Upjohn Co.,Kalamazoo, Mich.

3. Beachey, E. H., W. A. Simpson, and I. Ofek. 1980. Interaction ofsurface polymers of Streptococcus pyogenes with animal cells,p. 389-405. In P. Rutter and B. Vincent (ed.), Microbialadhesion to surfaces. Ellis Horwood Ltd., Chichester, England.

4. Courtney, H. S., W. A. Simpson, and E. H. Beachey. 1986.Relationship of critical micelle concentrations of bacterial

lipoteichoic acids to biological activities. Infect. Immun.51:414-418.

5. Dale, J. B., I. Ofek, and E. H. Beachey. 1980. Heterogeneity oftype-specific and cross-reactive antigenic determinants within asingle M protein of group A streptococci. J. Exp. Med.151:1026-1038.

6. Dean, E. A., and R. E. Isaacson. 1982. In vitro adhesion ofpiliated Escherichia coli to small intestinal villous epithelial cellsfrom rabbits and the identification of soluble 987P pilus recep-tor-containing fraction. Infect. Immun. 36:1192-1198.

7. Duguid, J. P., and R. R. Gillies. 1957. Fimbrial and adhesiveproperties in dysentery bacilli. J. Pathol. Bacteriol. 74:397-411.

8. Firon, N., I. Ofek, and N. Sharon. 1983. Carbohydrate speci-ficity of the surface lectins of Escherichia coli, Klebsiellapneumoniae, and Salmonella typhimurium. Carbohydr. Res.120:235-249.

9. Gaastra, W., and F. K. deGraff. 1982. Host-specific fimbrialadhesins of noninvasive enterotoxigenic Escherichia colistrains. Microbiol. Rev. 46:129-161.

10. Isaacson, R. E., P. C. Fusco, C. C. Brinton, and H. W. Moon.1978. In vitro adhesion of Escherichia coli to porcine smallintestinal epithelial cells: pili as adhesive factors. Infect. Im-mun. 21:392-397.

11. Jones, G. W., and R. E. Isaacson. 1984. Proteinaceous bacterialadhesins and their receptors. Crit. Rev. Microbiol. 10:229-260.

12. Korhonen, T. K., E. L. Nurmiaho, H. Ranta, and C. SvanborgEden. 1980. New method for isolation of immunologically purepili from Escherichia coli. Infect. Immun. 27:569-575.

13. Mackowiak, P. A., and M. Marling-Cason. 1984. A comparativeanalysis of in vitro assays of bacterial adherence. J. Microbiol.Methods 2:147-158.

14. Mirelman, D., and I. Ofek. 1985. Microbial lectins and aggluti-nins, p. 1-19. In D. Mirelman (ed.), Microbial lectins andagglutinins. John Wiley & Sons, Inc., New York.

15. Nagy, B., H. W. Moon, and R. E. Isaacson. 1977. Colonization ofporcine intestine by enterotoxigenic Escherichia coli: selectionof piliated forms in vivo, adhesion of piliated forms to epithelialcells in vitro, and incidence of a pilus antigen among porcineenteropathogenic E. coli. Infect. Immun. 16:344-352.

16. Ofek, I., and E. H. Beachey. 1978. Mannose binding andepithelial cell adherence of Escherichia coli. Infect. Immun.22:247-254.

17. Ofek, I., and E. H. Beachey. 1980. General concepts andprinciples of bacterial adherence in animals and man, p. 2-29. InE. H. Beachey (ed.), Bacterial adherence, series B, vol. 6.Receptors and recognition. Chapman & Hall, Ltd., London.

18. Stanislawski, L., W. A. Simpson, D. Hasty, N. Sharon, E. H.Beachey, and I. Ofek. 1985. Role of fibronectin in attachment ofStreptococcus pyogenes and Escherichia coli to human celllines and isolated oral epithelial cells. Infect. Immun.48:257-259.

19. Thorne, G. M., C. F. Deneke, and S. L. Gorbach. 1979.Hemagglutination and adhesiveness of toxigenic Escherichiacoli isolated from humans. Infect. Immun. 23:690-699.

20. Zilberberg, A., J. Goldhar, and I. Ofek. 1983. Adherence ofenterotoxigenic Escherichia coli (ETEC) strains to mouse intes-tines analysed by Langmuir adherence isotherms. FEMS Mi-crobiol. Lett. 16:225-228.

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