Vol. 32, No. 2INFECTION AND IMMUNITY, May 1981, p. 881-8880019-9567/81/050881-08$02.00/0

Attachment Factors Among Enterotoxigenic Escherichia colifrom Patients with Acute Diarrhea from Diverse Geographic

AreasMARK J. BERGMAN, WANDA S. UPDIKE, SHEILA J. WOOD, STUART E. BROWN, III, AND

RICHARD L. GUERRANT*Division of Geographic Medicine, Department ofMedicine, School ofMedicine, University of Virginia,

Charlottesville, Virginia 22908

To cause diarrhea, enterotoxigenic Escherichia coli (ETEC) must initiallycolonize the small bowel. Different surface structures have been implicated inthis initial attachment. Recognized attachment factors include colonization factorantigens I and II (CFA/I and CFA/II) and type I pili. Several methods ofdetection for each of these factors have been reported. In this study, we screenedfor the presence of these attachment factors among enterotoxigenic E. coliisolated from 40 patients with acute diarrhea and 40 asymptomatic controlindividuals and examined their ability to attach to ATCC 407 human intestinalcells in vitro. Of 40 patients with diarrhea, 16 (40%) had enterotoxigenic E. coliisolates which exhibited an attachment trait. Fourteen (35%) of these isolatesdemonstrated the ability to attach to ATCC 407 cells, whereas only four isolatesfrom asymptomatic controls attached (P < 0.02). A total of 20% of the patientisolates and 7.5% of the control isolates possessed CFA/I. Only one patient isolatedemonstrated CFA/II. Evidence for type I pili was found on 10% of the patientisolates and 12.5% of the control isolates. Attachment to ATCC 407 cells allowedthe detection of 87.5% (14 of 16) of enterotoxigenic E. coli isolates with any typeof attachment trait. Of the 14 cases demonstrating attachment ability to ATCC407 cells, 7 did not attach in the presence of mannose. Three of these showedevidence for both CFA/I and type I pili, one showed only CFA/I, and one showedonly type I pili. Two of those mannose-sensitive attaching isolates showed noother demonstrable trait. Seven patient isolates showed mannose-resistant at-tachment. Of these, two were classified as possessing CFA/I, and one was classifiedas possessing CFA/II. The four remaining isolates could not be classified into anyrecognized attachment factor category, suggesting that other attachment factorsremain to be identified.

In the pathogenesis of enterotoxigenic Esch-erichia coli (ETEC) diarrhea, initial coloniza-tion of the small intestine is necessary for theorganism to multiply there and produce suffi-cient quantities of enterotoxin to cause diarrhea(9, 11, 15, 21, 28). The ability of certain ETECto colonize has been ascribed to heat-labile pro-tein surface antigens, human colonization factorantigens (CFA/I and CFA/II) (5, 6, 8, 15, 25),porcine K88 antigen (15, 22), and bovine K99antigen (21, 28). These colonization factors ap-pear to be controlled by plasmids which can bespontaneously lost on subculture in the labora-tory (9, 19, 21, 28).Mannose-resistant hemagglutination

(MRHA) of human group A erythrocytes (6)may be a useful screening technique for thedetection of human CFA/I. Isolates that havelost CFA/I through subculture in the laboratory

do not exhibit MRHA. CFA/I has been detectedamong at least four different serogroups, includ-ing 015, 025, 063, and 078 (8). There is nowevidence that two other serogropus, 06 and 08,which are often associated with acute diarrheain humans, possess a different CFA antigen (5).This surface antigen (termed CFA/II) exhibitsMRHA of bovine erythrocytes but not humanerythrocytes.Another type of surface structure, the com-

mon type I pili, is readily visualized by electronmicroscopy and has also been implicated as animportant attachment factor ever since the orig-inal studies of Duguid et al. in 1955 (4). Similarstructures and their association with hemagglu-tinating and cell-adhering properties have beendescribed in Shigella flexneri and several Sal-monella species (2, 3). In contrast to CFA/I andCFA/II, the common type I pili do not aggluti-

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882 BERGMAN ET AL.

nate human erythrocytes but instead displaymannose-sensitive agglutination of guinea pigerythrocytes, which can be prevented by priorincubation of these E. coli organisms with man-nose (2-4, 18, 23, 24). Furthermore, Ofek et al.have demonstrated the elution of adherent E.coli to buccal epithelial cells by D-mannose ormethyl a-D-mannopyranoside (17). Type I-pi-liated E. coli can also be distinguished fromnonpiliated organisms by colonial morphologywhen grown on a nutrient agar (23, 24). E. colitype I pili are antigenically distinct from thepurified human CFA/I (20).We have described a simple in vitro assay for

attachment factors, using ATCC 407 human in-testinal cells in tissue culture (1).The number of distinct, recognizable surface

structures important for attachment of E. colito host cells and the number of assay techniquesfor these factors are both increasing; this studywas designed to compare the ability of an assaytechnique measuring the attachment of patientETEC isolates to ATCC 407 cells with the abilityof other assay techniques to detect attachmentfactors. We studied 80 E. coli isolates from 40patients with diarrhea and from 40 asympto-matic control individuals from the same popu-lations for adherence traits by the following cri-teria. We examined for attachment to ATCC 407cells in the presence and absence of mannose,MRHA of human type A erythrocytes and bo-vine erythrocytes, agglutination with antiserumagainst type I pili and CFA/I, and colonial mor-phology on agar plates. Attempts were also madeto block attachment with specific antisera, andfurther studies were done to explore the mech-anism of attachment of E. coli to ATCC 407cells.

MATERIALS AND METHODSBacterial cultures. A total of 40 ETEC isolates

from 40 patients with diarrhea and 40 nontoxigenicisolates from asymptomatic control individuals fromthe same populations were used in these studies. Ofthese, 12 case isolates were obtained from childrenwith diarrhea in Fortaleza, Brazil, in March throughMay 1978; 15 case isolates were obtained from adultswith acute diarrhea in Florianopolis, Brazil, in 1976; 7case isolates were obtained from Navajo adults andchildren with diarrhea at the Fort Defiance IndianHospital in 1975; and 6 case isolates were obtainedfrom patients with acute diarrhea in a family study inCharlottesville, Va., from 1975 to 1977. Control isolateswere from 10 asymptomatic individuals from each ofthe above four populations. Each isolate was testedfor production of the heat-stable and heat-labile en-terotoxins (ST and LT) by the previously describedsuckling mouse assay (10) and the Chinese hamsterovary cell assay (14), respectively.None of these strains demonstrated invasiveness by

the Sereny guinea pig conjunctivitis test (27).

INFECT. IMMUN.

For assay of attachment to human intestinal epi-thelial cells, E. coli isolates were cultured on CFAagar, (6), which consists of 1% Casamino Acids (DifcoLaboratories, Detroit, Mich.), 2% agar, and trace saltsat pH 7.4. This medium promotes the growth andformation of CFA antigens. Cultures were also grownon nutrient agar (BBL Microbiology Systems, Cock-eysville, Md.) for demonstration and isolation of E.coli with common type I phi (23, 24). Hemagglutina-tion and agglutination studies were performed on or-ganisms grown on CFA agar plates.

Tissue culture. ATCC 407 cells were obtainedfrom the American Type Culture Collection (CCL-6,human intestine 407) and were maintained in F-12medium (GIBCO Laboratories, Grand Island, N.Y.)with 10% fetal calf serum (GIBCO) as previously de-scribed (1). Bacterial attachment and morphologywere less consistent with ATCC 407 cells after eightor more laboratory passages. The morphology ofATCC 407 cells was therefore carefully monitored, andknown strains that were positive or negative for cellattachment were run as controls with each assay. Cellswere not used for adherence studies after eight pas-sages in our laboraotry.Assay for adherence of E. coli to ATCC 407 cell

monolayers. Modifications of previously describedmethods were used. At 24 h before assay, a suspensionof approximately 5 x 102 ATCC 407 cells in 0.3 ml ofantibiotic-free F-12 medium with 10% fetal calf serumwas added to each chamber of eight-chambered tissueculture slides (Lab-Tek Products, Naperville, Ill.). Themedium was again changed just before the bacteriawere added for assay. E. coli isolates grown 18 hovernight in CFA agar were suspended in phosphate-buffered saline (GIBCO) and added to each chamberat a final concentration of approximately 5 x 107bacteria per ml. In studies requiring mannose, 1%mannose in phosphate-buffered saline was used tosuspend the bacteria. Attachment with varyingamounts of mannose was compared, using 10, 1, 0.1,0.001, 0.0001, and 0.00001% mannose in phosphate-buffered saline as above. Cells were then incubatedwith bacteria for 3 h after which the slides werewashed three times with phosphate-buffered salinefixed with methanol, and stained with Giemsa stain.Adherence was considered positive if 20% or more ofthe cells had five or more cell-associated bacteria (Fig.1A) and negative if fewer than 20% of cells showedbacterial attachment (Fig. 1B). The ability of specificantisera to block adherence was studied by incubatingbacteria with a 1:8 dilution of antiserum before incu-bating them with ATCC 407 cells. At this dilution, novisible agglutination was seen. Antiserum preparedagainst purified type I pili was kindly supplied by I.Salit of the Rockefeller University (23). Anti-CFA/Iantiserum was kindly provided by D. G. Evans of theUniversity of Texas Medical School in Houston andwas used at a 1:8 dilution in phosphate-buffered saline(7). Serotyping was kindly performed by D. J. Evans,Jr., also of the University of Texas Medical School inHouston, and by D. J. Brenner at the Centers forDisease Control in Atlanta, Ga. Pretreatment ofATCC 407 cells with 0.6 U of neuraminidase (EC3.2.1.18; Sigma Chemical Co., St. Louis, Mo.) per mlwas done 1 h before assaying with a known CFA/I E.coli 078:H11.

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ENTEROTOXIGENIC E. COLI ATTACHMENT FACTORS 883

. ...

A BFIG. 1. ATCC 407 cells 3 h after incubation with CFA/I-positive (A) and CFA/I-negative (B) E. coli. Note

numerous CFA/I-positive E. coli attached to the cells in this Giemsa-stained preparation.

Hemagglutination. Hemagglutination studieswith human type A, bovine, and guinea pig erythro-cytes (Flow Laboratories, Inc., Rockville, Md.) werestudied by the method previously described (6). Vary-ing amounts of mannose were also studied, with logiodilutions ranging from 10% to 0.00001% mannose beingused as described above.

Bacterial agglutination with antiserum. Theslide antibody agglutination test was performed witheither anti-type I pili or anti-CFA/I antisera as pre-viously described (6).Colony morphology. Isolates were grown for 16

to 18 h on nutrient agar and segregated into colonytypes as previously described (23, 24).

Statistical evaluation. Results of the above testswere recorded as positive if that trait was present inthat isolate or as negative if that trait was absent.Statistical differences between traits of the case groupand controls were determined by chi-square analysis.

RESULTSTime course of attachment of E. coli to

HIE cells. The percentage of ATCC 407 cellswith >5 cell-associated E. coli (078:H11) atvarious incubation times is shown in Fig. 2.When there was careful control of ATCC 407cell morphology, attachment of CFA/I-positiveE. coli was noted at 1 h and was maximal at 3 h,when bacteria attached to 96.0 + 3.4% of the

100 F

80sPERCENT

ATCC 407 CELLSWITH >5BACTERIA

CELLSASSOCIATED

60 F

40

20

0.25 .5 1 hr 3 hrs. 5 hrs

TIME (hours)

FIG. 2. Time course of CFA/I E. coli attachmentto ATCC 407 cells. Points represent means ± stan-dard error of the mean.

ATCC 407 cells (different from what was previ-ously noted [1]). For this reason, the 3-h incu-bation time was used for the remainder of thestudy.Detection of attachment factors. Three

recognized types of attachment to human epi-

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884 BERGMAN ET AL.

thelial cells and methods of detection for eachtype are summarized in Table 1. Note that thereare several criteria, including agglutination inspecific antiserum and hemagglutination ofcharacteristic erythrocytes for these three rec-

ognized attachment traits (5, 6, 23, 24). Of the 16strains with evidence of one or more attachmenttrait, 8 exhibited some evidence for CFA/I, 1exhibited evidence for CFA/II, 4 exhibited evi-dence for type I pili (with a comparable numberamong control isolates also tested), and 14 dem-onstrated attachment to the human intestinalepithelial cells.CFA/I. There was not complete concordance

of results in different assay systems among theeight strains exhibiting some features of CFA/I.Whereas five isolates demonstrated both agglu-tination with specific anti-CFA/I antiserum andmRHA of human type A erythrocytes, twostrains exhibited only mRHA of type A eryth-rocytes, and one strain exhibited agglutinationonly in anti-CFA/I antiserum. Four of the fiveisolates with both features of CFA/I also at-tached to ATCC 407 cells, and this attachmentwas prevented by incubation of the bacteria withantiserum against CFA/I. However, this attach-ment was sensitive to mannose (in concentra-tions as low as 0.001%). Three of these strainsalso exhibited evidence for type I pili, but anti-serum against type I pili failed to prevent at-tachment to the ATCC 407 cells.

CFA/II. Only one case isolate demonstratedmRHA of bovine erythrocytes. This isolateshowed mannose-resistant attachment to ATCC407 cells.Type I pili. Four case isolates and five control

isolates were classified by all three criteria aspossessing the common type I pili. Seven ofthese nine isolates (all four case isolates) ex-hibited mannose-sensitive attachment to HIEcells; however, pretreatment with anti-type I piliantiserum did not block their attachment toATCC 407 cells. The remaining two isolates(from asymptomatic controls) also agglutinatedwith anti-CFA/I antiserum but did not attachand did not hemagglutinate bovine or humanerythrocytes.Attachment of E. coli to ATCC 407 cells.

Of 40 ETEC isolated from patients with diar-rhea, 14 (35%) demonstrated attachment to HIEcells. In contrast, only 4 of 40 (10%) nontoxigenicisolates from control individuals attached toATCC 407 cells (P < 0.02). Of the 14 case

isolates, 6 showed only attachment to the ATCC407 cells and did not exhibit evidence for CFA/I, CFA/II, or type I pili, suggesting that theattachment of these 6 case isolates to ATCC 407cells occurred by a different mechanism fromthe above recognized attachment traits. Bymeasuring attachment to ATCC 407 cells in theabsence of mannose, 14 of 16 (87.5%) of theattachment factors identified were detected. Thetwo isolates that did not attach to ATCC 407cells demonstrated agglutination in anti-CFA/Iantiserum, and one of these also demonstratedMRHA of human type A erythrocytes.Only 7 of the 14 case isolates (and 1 of the 4

control isolates) exhibited mannose-resistant at-tachment to ATCC 407 cells (Fig. 3). It shouldbe noted that the degree of attachment (per-centage of ATCC 407 cells with more than fiveE. coli attached) was less among the 10 strains

TABLE 1. Recognized attachment factors of E. coli

No. ofType of attachment Means of detection positive No. of positive con-

case iso- trol isolateslates

CFA/I Agglutination by anti-CFA/I antiserum 1 2MRHA of human A erythrocytes 2 1Both of above 5 0

CFA/II MRHA of bovine erythrocytes 1 0

Type I pilli Agglutination of anti-type I pili antiserum 4 5MRHA of guinea pig erythrocytes 4 5Rounded, glistening colonies 4" 5Attachment to ATCC 407 cells only 6 lb

Attachment to ATCC 407 cells + another 8 3htrait

Total with one or more 16 6 (P < 0.03)attachment traits

a Of these 4 strains, three also exhibited both CFA/I traits as well as mannose-sensitive attachment to ATCC407 cells.

b In contrast to 14 isolates from cases, a total of four isolates from control individuals attached to ATCC 407cells (P < 0.02).

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ENTEROTOXIGENIC E. COLI ATTACHMENT FACTORS 885

(7 case and 3 control isolates) thatmannose-sensitive attachment to Acells. Of the seven case isolates thatmannose-resistant attachment (Tabl4two showed evidence of CFA/I with]human type A erythrocytes. One demMRHA of bovine erthrocytes, suggEpresence of CFA/II, and four isolatEother demonstrable traits. Of the sevdemonstrating mannose-sensitive atta(the ATCC 407 cells, three had evidencCFA/I and type I pili. However, thement to the ATCC 407 cells was blockeCFA/I antiserum but not by anti-tiantiserum suggesting that their attacthese cells involved CFA/I, even thoi

,00

soPERCENT

ATCC 407 CELLSWITH >5BACTERIA 60

CELLSASSOCIATED

40

20

ATTACHUEN

_ ATTACHMEW

n 3

Cass Controh Case controisMaennose - ts'stent Mnomo"- Sensitime

AttGchnent Attochmnnt

(A, (A, )

FIG. 3. Comparison of case and contisolate attachment to ATCC 407 cells afterof the bacteria with or without 1% manalso the more extensive attachment of thresistant isolates.

TABLE 2. Other characteristics of 14 ET]that attached to ATCC 407 cell

Atta

Attachment characteristic Mannoseresistant

MRHA of:Human A erythrocytes 2Bovine erythrocytes 1

Agglutination in anti- 0CFA/I antiserum

Type I pili characteristics 0Attachment to ATCC 407 4

cells only

Total no. of isolates 7

a Of these seven mannose-sensitive ET]three also showed MRHA of human A eragglutination in anti-CFA/I antiserum, ancistics of type I pili; one also showed botbhuman A erythrocytes and agglutination irI antiserum.

exhibited mannose-sensitive. One strain each of those thatTCC 407 demonstrated mannose-sensitive attachment toexhibited ATCC 407 cells demonstrated evidence only fore 2), only CFA/I or type I pili, respectively. The two otherMRHA of strains that demonstrated mannose-sensitive at-onstrated tachment to ATCC 407 cells did not exhibit theasting the secondary evidence of CFA/I, CFA/II, or typeas had no I pili, for which we examined.en strains Data regarding the 16 E. coli case isolateschment to possessing at least one attachment trait are'e for both shown in Table 3. Nine produced ST, five pro-,ir attach- duced LT, and two produced both LT and ST.dby anti- Of the nine ST producers, six demonstratedype I pili mannose-sensitive attachment to ATCC 407-hment to cells, whereas none of the five LT strains showedagh it was this characteristic. Of the remaining three ST

isolates, one showed characteristics of CFA/Ibut did not attach to ATCC 407 cells, one

WITHWOUT MANNOSE showed CFA/II and mannose-resistant attach-ITWITH INNOSE ment to ATCC 407 cells, and the last showed

mannose-resistant attachment, but no recog-nized attachment trait was demonstrated. Allfive LT isolates showed mannose-resistant at-tachment to ATCC 407 cells. Of these, twoshowed partial evidence for CFA/I (MRHAonly). The other three LT isolates showed noother attachment factors. Two isolates producedboth ST and LT, one showed mannose-sensitive

^|2- g3- attachment to ATCC 407 cells and demon-Cas controls strated CFA/I and type I pili, and one showedNoAttachment no attachment to ATCC 407 cells but exhibited

CFA/I.trol E. coli Mannose-sensitive attachment with var-incubation ied mannose concentrations. Attachment ofnose. Note the seven case isolates demonstrating mannose-e mannose- sensitive attachment was studied with different

concentrations of mannose. Incubation of theseEC isolates organisms with mannose at concentrations of 1,s 0.1, 0.001, 0.0001, and 0.00001% are shown in Fig.chment 4. There is significant inhibition of attachment

at mannose concentration of 0.001% or greater- Mannose- (P < 0.003); however, there is no significant

sensitivea effect at 0.00001% mannose.MRHA of human type A erythrocytes at

4 various mannose concentrations. The seven0 case isolates demonstrating MRHA with human

type A erythrocytes were studied with increasingmannose concentrations. All seven of these iso-lates retained their MRHA at mannose concen-

2 trations of 0.1, 1, and 10%.Attachment of case isolates to neuramin-

idase-treated ATCC 407 cells. Because sialic7a acid reduced the adherence of CFA/I-positive

EC isolates, E. coli to ATCC 407 cells (1, 13), we examinedrythrocytes, the effect of neuraminidase treatment of thecharacYter- cells on attachment of the E. coli case isolates.d

MRHA of To examine the potential role of neuraminidase-n anti-CFA/ sensitive sialic acid residues as possible receptors

for E. coli attachment factors, attachment of

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TABLE 3. Enterotoxin production and attachment factors'Attachment factorEnterotoxin production (no. of iso- Isolate no. Serotype

lates) CFA/I CFA/II Type I pili

STAttachment to ATCC 407 cellsMannose sensitive (n = 6) C1 049:H12 + - +

C1l 078:H12 + - +F39 NT + - -N48 - +F9 NT - - -F33 NT - - -

Mannose resistant (n = 2) C24 092:NM - +C27 015:H18 - - -

No attachment to ATCC 407 N39 + (agglutinationcells (n = 1) only)

LTNo mannose-sensitive

attachment to ATCC 407cells

Mannose-resistant attachmentto ATCC 407 (n = 5) NA8 NT + (MRHA only)

Si O1:H- + (MRHA only)F22 NT - - -F24 NT - - -Kl

LT + STMannose-sensitive attachment Fl1 NT + - +

to ATCC 407 cellsNo attachment to ATCC 407 FIO 063:NM +

a Sixteen ETEC isolates with evidence for an adherence trait were tested. C and F, Isolates from northeasternand southern Brazil, respectively; N, isolate from Fort Defiance; S and K, isolates from Charlottesville. NT,Nontypable.

2624

22

20PERCENT 8

ATCC 407 CELLSWITH>5 16BACTERIA 14CELLASSOCIATED 2

10

8

64

2-

so

PERCENTATCC 407 CELLS

WITH >5BACTERIA

CELLSASSOCIATED

0 -- --2 -3 -4- NOMANNOSE

LOG % MANNOSE CONCENTRATIONFIG. 4. Attachment ofeach E. coli isolate to ATCC

407 cells at varied mannose concentrations (log per-cent). Note the inhibition of attachment at mannoseconcentrations of 0.001% or higher (P < 0.003),whereas there is no significant effect at 0.00001%mannose (P = 0.27).

case isolates to ATCC 407 cells after the cellswere treated with 0.6 U of neuraminidase per mlwas examined. There was no reduction of theattachment of any of the case isolates by neur-aminidase (Fig. 5). Mannose-resistant E. coli

n£ 7

JI UNTREATED ATCC 407 CELLS

_ NEURAMINIDASE-TREATED ATCC 407 CELLS

n * 7

'.

Mannose -Resstent Monnose-USnsiLoveAttochment At?ach,,,mmt

No Attachment

FIG. 5. Comparison of the attachment of case E.coli isolates to ATCC 407 cells before and after in-cubation of the cells with 0.6 U ofneuraminidase perml.

attached equally well to neuraminidase-un-treated and -treated ATCC 407 cells (91.6 + 5.0and 93.2 + 5.1%, respectively). The attachmentof the seven mannose-sensitive E. coli appearedto increase after ATCC 407 cells were treatedwith neuraminidase. The organisms that did not

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ENTEROTOXIGENIC E. COLI ATTACHMENT FACTORS 887

attach to untreated ATCC 407 cells also failedto attach to neuraminidase-treated cells.

DISCUSSIONSeveral models have recently been introduced

that assay for different attachment and coloni-zation factors: MRHA of human type A eryth-rocytes for CFA/I (6); mannose-sensitive hem-agglutination of guinea pig erythrocytes and co-lonial morphology for the common type I pili(23, 24); and in vitro bacterial attachment toATCC 407 cells (1). Recent data from humanvolunteer studies (8, 25) showed that ETECstrains differing in the presence or absence ofthe plasmid coding for CFA/I differ in theirability to cause diarrhea. Organisms that possessthe CFA/I antigen consistently cause diarrhea,whereas identical strains without CFA/I do not.Results from another study showed that ETECorganisms possessing CFA/I were isolated in86% of cases of 29 adults with diarrhea acquiredin Mexico and in only 18% of the asymptomaticcontrols (7). However, Gross et al. (12) foundthat of 89 ETEC strains isolated from patientswith diarrhea, only 10% showed MRHA and 7%possessed the CFA/I antigen. In another studyof six ETEC strains isolated from patients withdiarrhea, only one strain showed MRHA of hu-man group A erythrocytes and agglutinated withanti-CFA/I antiserum (16). Four other strains inthat study demonstrated mannose-sensitivehemagglutination. All six ETEC strains wereclearly shown to colonize and cause diarrhea inhuman volunteers.Our study explored several types of attach-

ment factors among 40 ETEC case isolates and40 control isolates from four different geographicareas. We compared assay techniques for CFA/I, CFA/II, and type I pili (5, 6, 23) with an assaymeasuring attachment to the ATCC 407 cells intissue culture. Of the 40 case isolates, 16 (40%)demonstrated at least one of the eight attach-ment traits studied. Of these 16, 8 exhibitedevidence for CFA/I, one could be classified aspossessing CFA/II, and four demonstrated evi-dence for type I pili (Table 1). In six cases, theisolates only demonstrated attachment to ATCC407 cells. Evidence for type I pili was demon-strated in 10% of the case isolates and 12.5% ofthe control isolates. Although antigenic variabil-ity or structural microheterogeneity of type I pilicould explain the failure of anti-type I pilusantisera to block attachment to these cells, ourdata do not support a major role for type I pilias adherence traits among ETEC.Of the 16 case isolates demonstrating at least

one attachment trait, 14 (87.5%) showed attach-ment to ATCC 407 cells in vitro; only 2 caseisolates (that possessed CFA/I) failed to attach.

Initial screening for adherence to ATCC 407cells, therefore, would identify the majority ofattachment traits found by other methods aswell as a significant number (6, or 15%) of otherisolates that appeared to be adhering by previ-ously unrecognized attachment traits. Sincemultiple attachment factors appear to be asso-ciated with colonization of the small bowel byETEC, this type of functional bioassay may beof value as a screening test. Initial screening forattachment to ATCC 407 cells appears to detectmost previously recognized attachment factorsas well as additional colonizing traits that arenot yet well characterized. There was no partic-ular pattern of serotype or of geographic originwith typed adherence traits (Table 3). ElevenETEC isolates with adherence traits were fromnortheastern or southern Brazil, three were fromFort Defiance, and two were fron Charlottes-ville.

Bacterial attachment to the ATCC 407 cellswas studied after 3 h instead of 5 h of incubation,as reported previously (1), since attachment ofa known CFA/I-positive E. coli isolate was max-imal at 3 h when the morphology of the cells wascarefully monitored and when they were notused after eight laboratory passages. Further-more, recent observations in our laboratory onthe attachment of known CFA/I-positive E. colito other human and nonhuman tissue culturecells have shown species-specific attachment tohuman cell lines intestine 407 (ATCC 407 CCL-6), cervix (HeLa, CCL-2), and liver (Chang,CCL-13), but not to canine kidney (MDCK,CCL-34) or hamster ovary (CHO-Kl, CCL-61)cells (unpublished data). Further studies on thisspecies specificity are in progress.Much remains to be learned about the mech-

anism of ETEC attachment to the small boweland the nature of the attachment receptor(s).From our finding that the attachment of fourCFA/I-positive ETEC isolates to ATCC 407cells was mannose sensitive, it appears thatCFA/I attachment to different cells is variablyaffected by mannose, and receptors on differentcells for CFA/I appear to differ. Although CFA/I attachment to ATCC 407 cells had been shownto be inhibited by N-acetyl neuraminic acid (1),neuraminidase treatment increased rather thandecreased adherence ofETEC to the cells. Thus,stripping the cell surface of N-acetyl neuraminicacid may reduce the surface charge density andallow closer approximation of attachment fac-tors to receptors (26).

In conclusion, it is evident that there are mul-tiple factors involved in the initial attachmentand subsequent colonization of the small intes-tine by ETEC. Besides the recognized attach-ment factors, there appear to be additional traitsthat have yet to be characterized. Attachment

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888 BERGMAN ET AL.

to ATCC 407 cells in tissue cultures was signifi-cantly associated with diarrhea case (versus con-trol) isolates, and the assay measuring this at-tachment was the most sensitive single assay forattachment traits. It is also evident that man-nose sensitivity of adherence varies with the celltype being examined. Finally, the specific recep-tors in intestinal tissue for these attachmentfactors and their mechanism of adherence re-main to be defined.

ACKNOWLEDGMENT

This work was supported in part by Public Health Servicecontract N01-AI-42548 from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED

1. Bergman, M. I., D. G. Evans, G. L. Mandell, J. A.Sullivan, I. E. Salit, and R. L. Guerrant. 1978. At-tachment of Escherichia coli to human intestinal epi-thelial cells: a functional in vitro test for intestinalcolonization factor. Trans. Assoc. Am. Physician 91:80-89.

2. Duguid, J. P., E. S. Anderson, and I. Campbell. 1966.Fimbriae and adhesive properties in Salmonellae. J.Pathol. Bacteriol. 92:107-138.

3. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae andadhesive properties in dysentery bacilli. J. Pathol. Bac-teriol. 74:397-411.

4. Duguid, J. P., I. W. Smith, G. Dempster, and P. N.Edmunds. 1955. Non-flagellar filamentous appendages("fimbriae") and haemagglutinating activity in Bacte-rium coli. J. Pathol Bacteriol. 70:335.

5. Evans, D. G., and D. J. Evans, Jr. 1978. New surface-associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli ofserogroups 06 and 08. Infect. Immun. 21:638-647.

6. Evans, D. G., D. J. Evans, Jr., and W. Tjoa. 1977.Hemagglutination of human group A erythrocytes byenterotoxigenic Escherichia coli isolated from adultswith diarrhea: correlation with colonization factor. In-fect. Immun. 18:330-337.

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