Colonization Factors of Enterotoxigenic Escherichia coli ... - Europe PMC

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1991, p. 1893-1898

Vol. 29, No. 9

0095-1137/91/091893-06$02.00/0 Copyright (C 1991, American Society for Microbiology

Colonization Factors of Enterotoxigenic Escherichia coli Isolated from Children with Diarrhea in Argentina NORMA BINSZTEIN,l* MABEL J. JOUVE,l GLORIA I. VIBOUD,1 LAURA LOPEZ MORAL,' MARTA RIVAS,' IDA 0RSKOV,2 CHRISTINA AHREN,3 AND ANN-MART SVENNERHOLM3 Instituto Nacional de Microbiologia "Carlos G. Malbran," Velez Sarsfield 563, 1281 Buenos Aires, Argentina1; International Escherichia and Klebsiella Centre, Statens Seruminstitut, 2300 Copenhagen, Denmark7; and Department of Medical Microbiology and Immunology, University of Goteborg, S41346 Goteborg, Sweden3 Received 31 December 1990/Accepted 31 May 1991

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and the E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes 0153:H45 and 078:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype 0128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the 06:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistent hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.

not cause MRHA (38). Enterotoxin biosynthesis is not associated with CS4 or CS6 expression (41). CFA/I and CFA/II have been shown to have strong bacterial surface hydrophobicity (16), but this characteristic has not been studied for CFA/IV-positive strains. CFA-carrying ETEC are capable of inducing protective immunity (3, 19) and are candidates for inclusion in a multivalent vaccine (32). Therefore it is important to know the distribution of the CFAs in ETEC strains in different areas of the world and also to search for possible new adhesive factors. Some surveys have been carried out to determine the prevalence of CFA/I, CFA/II, and CFA/IV in ETEC strains in different countries, and the reported results have varied between 25 and 75% (7, 13, 21, 27, 39). The aim of the present study was to study CFAs of ETEC in Argentina. This was done by examining prospectively the expression of CFAs on 109 ETEC strains isolated from 1,211 children with diarrhea during a period of 18 months. Monoclonal antibody (MAb)-based immunological methods were used to look for CFA/I, CFA/II, and CFA/IV and their CS subcomponents. The relationship between the expression of CFAs and production of enterotoxins, MRHA pattern, hydrophobicity, and serotypes was also studied.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of acute diarrhea in children in developing countries and in travelers to those countries (4). ETEC have two major virulence determinants: toxins (heat labile [LT] and heat stable [ST] enterotoxins) and colonization factor antigens (CFA). Both determinants are probably required for bacteria to cause disease. The colonization factors in human ETEC strains are usually protein fimbriae, which are capable of agglutinating erythrocytes from different animal species in the presence of D-mannose. The most well characterized of the CFAs are CFA/I, CFA/II, and CFA/IV (formerly called PCF 8775) (9, 10, 37). A close relationship between these CFAs and particular serotypes has been reported (6, 27, 39). In recent years several additional putative colonization factors have been described in ETEC strains that are associated with restricted serogroups: e.g., CFA/III (15), PCF0159 (36), PCF0148 (17), PCF0166 (25), PCFO9 (14), and CS17 (26). CFA/I is a single fimbrial antigen, whereas CFA/II and CFA/IV consist of several subcomponents. These components are called E. coli surface (CS) antigens; CFA/II comprises the CS1, CS2, and CS3 (8, 31) antigens, and CFA/IV comprises the CS4, CS5, and CS6 antigens (38). CFA/I causes mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes (11) and is controlled by a plasmid that also encodes production of ST (42). CFA/II causes MRHA of bovine but not human erythrocytes (11), and genes encoding CFA/II expression have been associated with plasmids coding for both LT and ST (30). CS4- and CS5-carrying strains have been reported to agglutinate human group A and bovine erythrocytes, whereas CS6 does *

MATERIALS AND METHODS Bacterial strains and culture conditions. A total of 109 ETEC strains isolated from 1,211 cases of diarrhea in children under 4 years of age were studied. The E. coli strains had been isolated during an 18-month period. The samples were collected from outpatient or hospitalized children at seven health centers situated in different areas of Argentina. Fecal specimens were collected from all children shortly after arrival in the health center and immediately

Corresponding author. 1893

1894

BINSZTEIN ET AL.

cultured on MacConkey agar plates for selection of E. coli isolates. After overnight culture, five E. coli colonies from each patient were incubated in individual deep agar vials and then sent to Buenos Aires for toxin and CFA analyses. Immediately after arrival in Buenos Aires, the bacteria were subcultured in Casamino Acids medium containing lincomycin and glucose for enterotoxin testing. The E. coli isolates identified as enterotoxigenic were maintained at -70°C in Trypticase soy broth (TSB; Difco, Detroit, Mich.) supplemented with 15% glycerol. Immediately before CFA analyses, all strains were retested for enterotoxin production, since it is well known that loss of enterotoxicity usually is associated with loss of CFAs; strains that did not produce any enterotoxin at the time of these analyses were omitted from the study. The ETEC strains to be tested for CFAs were inoculated directly from the -70°C vials into TSB and grown for 4 h without shaking. Each culture was then streaked on Casamino Acids-yeast extract agar (CFA agar) (10) with or without bile salts (25) and incubated at 37°C overnight. The bacterial confluent growth was suspended in 0.85% NaCl to a concentration of -101o CFU/ml. In all assays the following reference strains were included: H10407 (078:K80:H11, CFA/I, LT and ST), kindly provided by D. G. Evans, Houston, Tex.; and E1392-75 (06:K15: H16, CS1 and CS3, LT and ST), 58R957 (06:H16, CS2), C916-82 (025:H42, CFA/IV, CS4 and CS6, LT and ST), E11881A (025:H42, CS4 and CS6, LT and ST), and E17018A (0167:H22, CS5 and CS6, LT and ST), kindly provided by B. Rowe, London. Antibody preparations. Adsorbed rabbit antisera to CFA/I and CFA/II were prepared in Argentina as previously described (2). Adsorbed antisera to CFA/IV were produced by immunizing rabbits with strain E11881A (CS4 and CS6) or E17018A (CS5 and CS6) and adsorbing with the corresponding CS-deficient mutants (33). MAbs against CFA/I and the different CS components of CFA/II and CFA/IV were prepared as previously described (22, 24). The following MAbs were used: CFA/I 1:6, CS1 6:1, CS2 10:3, CS3 10:2, CS4 4:6, and CS5 5-1:1. Enterotoxin test. The 109 strains were analyzed for the production of LT and ST by means of ganglioside GM1enzyme-linked immunosorbent assays (GM1-ELISAs) (34, 35) with direct culture in plates (33). HA. Fresh human type A, bovine, guinea pig, chicken, goat, sheep, rabbit, and horse erythrocytes were obtained, washed twice in 0.85% NaCl, and suspended to 3% in saline with or without 1% D-mannose. Samples of 10 [L of the different erythrocyte suspensions were mixed with 10 [lI of the bacterial suspensions and observed for 2 min at room temperature and for another 5 min at 4°C when bovine erythrocytes were used. The hemagglutination (HA) was considered to be MRHA if the same degree of HA occurred with and without D-mannose (11). SO test. The salting out (SO) test was performed essentially as described by Lindahl et al. (20). A sample of 10 pul of the bacterial suspension was mixed on a glass slide with an equal volume of different concentrations of an ammonium sulfate solution. The mixture was rotated for 2 min at room temperature, and bacterial aggregation was observed. The bacterial surface hydrophobicity was measured by determining the lowest concentration of ammonium sulfate in which bacteria aggregated. Serum agglutination test. A 10-pA sample of bacteria (about 1010 organisms per ml) was mixed with 10 pI of nondiluted CFA antiserum on a glass slide. The appearance of macro-

J. CLIN. MICROBIOL.

scopically visible agglutinates within 2 min was regarded as a positive reaction. Bacteria mixed with saline were used as negative controls. Immunodiffusion tests. Crude antigens for immunodiffusion analyses were prepared as previously described (13, 27). The different antigen preparations were analyzed for CFA content by means of comparative immunodiffusion analyses (28) with a microplate modification of the original Ouchterlony test (40). Crude CFA/I and the subcomponents of CFA/II and CFA/IV antigens were prepared from the corresponding above-mentioned reference strains and used as reference antigens. Inhibition ELISA. In some instances the CFA properties were also analyzed by means of inhibition ELISA tests (22, 24) in which the capacity of ETEC to inhibit binding of MAbs against CFA and CS factors to corresponding solidphase bound purified CFA was assessed. Dot blot test. A modification of the nitrocellulose replica method (23) was in some cases also used for identification of CFAs. Nitrocellulose filter paper (HAWP, 045-pum pore size; Millipore Corp., Bedford, Mass.) was soaked in saline solution. Then 2 p.l of crude immunodiffusion antigen was applied to the filter paper and left at 37°C for 60 min. The paper was washed several times with gentle agitation. CFAcarrying preparations were visualized by using an ELISA (23) with specific MAbs. In each test appropriate positive and negative control strains were included. Only dots that were clearly stained were regarded as positive. Serotyping. The ETEC strains were serotyped at the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark. Testing procedure. From each child three E. coli colonies that had tested as enterotoxin positive immediately before CFA analyses were analyzed for the expression of CFAs. Each colony was tested for CFAs according to the following scheme: the strains, kept at -70°C, were grown for 4 h in TSB at 37°C. Each culture was streaked on CFA agar with and without bile salts (0.5%) and incubated overnight at 37°C. The cultures were suspended in saline and tested for MRHA and SO. Bacterial suspensions were also tested by slide agglutination with anti-CFA/I and anti-CFA/II (CS1 and CS3) polyclonal antisera and with anti-CS5 MAb in the slide agglutination assay and/or the inhibition ELISA. Strains that agglutinated with the polyclonal anti-CFA/I serum were similarly tested with the corresponding MAb, to confirm the CFA/I properties. Likewise, strains that agglutinated with the CFA/II polyclonal antiserum were also tested with anti-CS1 and anti-CS2 MAbs. The anti-CS3 MAbs available (24) were not used in slide agglutination, since they usually agglutinated strains positive for CS1 and CS3 and for CS2 and CS3 poorly. Instead, CFA/II-positive strains that were negative for CS1 and CS2 were tested for the presence of CS3 in the inhibition ELISA. Slide agglutination could not be used to search for CS4 and CS6 factors, since CS4-positive strains usually agglutinate spontaneously and CS6 antibodies do not agglutinate bacteria expressing this factor. Therefore, all strains that did not react with CFA/I, CFA/II, or CS5 antibodies in slide agglutination were tested for reactivity with anti-CFA/IV (CS4 and CS6) serum in immunodiffusion. Strains that were positive in immunodiffusion with CFA/IV antiserum but negative for CS4 were tested with the inhibition ELISA or dot blot test for CS5. Furthermore, all autoagglutinating strains were tested for the expression of either of the CFAs in immunodiffusion analyses. If one or more of the three colonies tested from each child gave similar results in enterotoxin and CFA analyses

VOL. 29, 1991

ETEC COLONIZATION FACTORS IN ARGENTINA

TABLE 1. Presence of CFA/I, CFA/II, and CFA/IV in relation to toxin production in ETEC strains isolated from children with diarrhea in Argentina ST

CFA/I CFA/II CS1-CS3 CS2-CS3 CS3 CFA/IV CS4-CS6 CS5-CS6 CS6 None Total

LT

LT and ST

25

0

0

1 0 0

0 0 0

10 1 1

0 16 3 26 71 (65)

0 0 0 23 23 (21)

0 0 0 3 15 (14)

TABLE 2. MRHA of CFA-positive and CFA-negative E. coli strains CFA property

No. (%) of strains producing toxin

Marker

Total

25 13 11 1 1 19 0 16 3 52 109

(23) (12)

(17)

(48)

and belonged to the same serotype, they were regarded as the same ETEC strain. In the case of differences between the individual colonies the child was said to be infected with more than one ETEC strain.

1895

No. of strains

CFA/I CFA/II CS5-CS6

CS6

CFA negative

MRHA of erythrocytes' Hu

Bv

Sh

Gp

Ch

Go

25 13 6 6 2 2 1 1

+ -

+ +

NTb

-

NT NT

-

-

-

_

NT NT

-

NT +

_

+

+ -

+ +

+ +

-

-

_ _ _

-

-

-

-

-

-

-

-

+

1

-

+

-

_

_ _

_ _

+ +

+

_

7 7 2 2 2 4C

-

-

-

-

-

-

+ _

-

+

-

-

+

-

-

_

_

-

-

-

+

+

_

a Hu, human blood group A; Bv, bovine; Sh, sheep; Gp, guinea pig; Ch, chicken; Go, goat. bNT, not tested. c These four strains had different HA patterns.

RESULTS Relationship between production of CFAs and enterotoxigenicity. One hundred nine ETEC strains were isolated from 1,211 episodes of infantile diarrhea as part of a multicenter survey that has been carried out in Argentina; 71 E. coli isolates produced ST only, 23 isolates produced LT only, and 15 isolates produced both LT and ST (Table 1). Of the 109 isolates, 57 (52%) carried one of the CFAs tested: i.e., 25 (23%) strains expressed CFA/I, 13 (12%) strains expressed CFA/II, and 19 (17%) strains expressed CFA/IV. Whereas all CFA/I and CFA/IV strains produced ST alone, 12 of the 13 CFA/II strains produced ST and LT. In four children, two different ETEC strains that also belonged to distinct serotypes were isolated. In two of these four cases, differences in CFA expression were found. CFA analyses. (i) CFAII. All of the 25 strains that agglutinated human and bovine erythrocytes (Table 2) reacted with both the polyclonal anti-CFA/I serum and the anti-CFA/I MAb, 1:6. All of the 25 CFA/I strains also possessed high hydrophobicity properties, i.e., they aggregated in ammonium sulfate in concentrations between 0.01 and 0.06 M. The prevailing serotypes of the CFA/I-positive ETEC were 0153:H45 and 078:H12 (Table 3). (ii) CFA/H. Fourteen of the ETEC strains only hemagglutinated bovine erythrocytes (Table 2). Thirteen of these strains were classified as CFAIII positive; 11 of them were identified as CS1-CS3 positive, 1 was identified as CS2-CS3 positive by agglutination with the respective polyclonal antibodies and MAbs, and the remaining strain was identified as CS3 positive as shown by immunodiffusion tests (Table 1). All the CFA/II-positive ETEC strains were also aggregated by ammonium sulfate, although the ammonium sulfate concentrations (0.08 to 0.8 M) needed were higher than those needed to agglutinate CFA/I-positive strains. Among the CS1-CS3-positive strains, 06:K15:H16 was the predominant serotype (10 of 11), the CS2-CS3-positive strain belonged to serotype 06:K15:H-, and the strain that was positive for CS3 only was 08:K40:H9 (Table 3). (iii) CFA/IV. Nineteen ETEC strains gave a precipitation line corresponding to the CS6 antigen when they were tested with anti-CFA/IV antiserum in the immunodiffusion test. Of

these 19 strains, 16 also gave a positive reaction in slide agglutination or in a dot blot test when an anti-CS5 MAb was used; accordingly, these strains were designated as CS5-CS6 positive. The remaining three strains expressed CS6 alone. No strain expressing CS4 was identified (Table 1). Ten of the 16 (62.5%) CS5-CS6-positive ETEC strains gave MRHA with sheep erythrocytes when they were grown in presence of bile salts (Table 2), whereas the strains positive for CS6 only did not show a defined HA pattern. Seventy-five percent of the CS5-CS6-positive ETEC strains were of serotype 0128:H21, and two of the three CS6positive strains were of the 0148:H28 serotype (Table 3). CFA-negative strains. Of the 109 ETEC isolates tested, 52 (48%) could not be identified as positive for CFA/I, CFA/II, or CFA/IV. Among these strains, some gave MRHA and/or SO (Table 4). Of these 52 ETEC strains, 24 produced MRHA with any of the erythrocytes tested, except with horse and rabbit erythrocytes, which always gave MRHA-negative results; strains that gave MRHA of chicken and guinea pig erythrocytes predominated (Table 2). Among the chicken MRHA-positive ETEC strains, five belonged to the 020:K+: H30 serotype, whereas those that gave MRHA of guinea pig TABLE 3. Serotypes in CFA-positive ETEC strains Colonization factor

Enterotoxin(s)

Total no. of strains

CFA/I

ST

25

CS1-CS3 CS2-CS3 CS3 CS5-CS6

LT, ST ST LT, ST LT, ST ST

10 1 1 1 16

CS6

ST

3

Serotype (no. of strains)

0153:H45 (12) 078:H12 (9) 078:H- (1) 0153:H- (1), 085:H4 (1) Rough:H45 (1) 06:K15:H16 (10) 086:H18 (1) 06:K15:H- (1) 08:K40:H9 (1) 0128:H21 (12), 029:H21 (2) Rough:H21 (1), 0128ac:H21 (1) 0148:H28 (2), 027:H7 (1)

1896

J. CLIN. MICROBIOL.

BINSZTEIN ET AL.

TABLE 4. MRHA and SO properties of strains No. of strains producing toxin

Properties

MRHA positive,a SO positiveb MRHA positive, SO negative MRHA negative, SO positive MRHA negative, SO negative

ST

LT

LT and ST

Total

0 16 1 9

3 4 1 15

0 1 2 0

3 21 4 24

a MRHA of human, bovine, guinea-pig, sheep, chicken, and/or goat erythrocytes. b SO, aggregation in c2 M ammonium sulfate.

erythrocytes belonged to seven different serotypes. Fortyfive strains were not able to aggregate in 2 M ammonium sulfate (Table 4). DISCUSSION

Previous surveys of ETEC strains in various parts of the world have shown wide variations in the number of E. coli strains with adhesion factors. In a study of ETEC isolated from clinical cases in Bangladesh (13), 75% of the strains expressed CFA/I or CFA/II; on the other hand, only 32.5% of the ETEC strains isolated from various categories of patients in South East Asia (39) possessed CFA/I, CFA/II, or CFA/IV and 29% of the ETEC strains collected from Thai patients expressed these CFAs (6). With regard to Latin America, 68% of the ETEC isolates were identified as CFA/I positive and 11% were identified as CFA/II positive in a study in Chile (1), whereas CFA/I and CFA/II were only found in 23% of the ETEC strains isolated in Brazil (29). As far as we know the present report is the first one describing the occurrence of colonization factors in ETEC in Argentina and the first analysis of CFA/I, CFA/II, and CFA/IV and their components in South America. In this survey of CFAs in ETEC isolated from seven hospitals in Argentina, CFA/I, CFA/II, and/or CFA/IV was produced by 52% of the ETEC isolates. CFA/I, the most prevalent adhesin antigen, was expressed by 23% of the strains, whereas CFA/IV and CFA/II were produced by 17 and 12% of the strains, respectively. In our study, methods with different specificities and sensitivities were used. The initial screening with relatively nonspecific reagents was followed by confirmation with highly specific MAb-based tests. The difference between our results and those mentioned above may be due to differences in the expression of CFAs on ETEC in various geographic areas or to the use of different laboratory techniques for the identification of the CFAs and enterotoxins. On the other hand, in Mexico Lopez-Vidal et al. (21), using essentially the same methods as those in the present study, have shown almost the same proportion (50%) of CFA-positive strains. The low percentages of strains carrying CFAs and enterotoxins in certain areas may also be due to repeated subculturing of strains and loss of plasmids. Surveys of ETEC have shown that each colonization factor is associated with particular serogroups and enterotoxin types. CFA/I has been associated with ST-positive and ST-LT-positive E. coli strains of restricted serotypes. Our results are in agreement with those in a survey carried out in Chile (1) in which CFA/I was only found in ST-positive ETEC strains. In Brazil (29), on the other hand, CFA/I was found in both ST-LT-positive and ST-positive strains. The reason for this discrepancy may be related to the prevalent CFA/I ETEC serotype found in

each study; i.e., serotypes 063:H- (often associated with LT-ST-positive strains) and 0128:H12 were found in Brazil, whereas serotype 0153:H45 was found in Chile and serotypes 0153:H45 and 078:H12 (generally associated with ST-positive ETEC) were found in Argentina. The serotype 0153:H45 was previously suggested to be a special SpanishLatin American type (5) because of its common occurrence in Spain and South America (1, 5). In our study, a CFA/Ipositive ETEC strain belonging to the serotype 085:H4 was found for the first time. Within the group of CFA/II-positive strains, we found that the ones containing CS1 and CS3 predominated over the strains containing CS2 and CS3 or CS3 only. Even though most of the CFA/II-positive strains produced LT and ST, as previously described by others (27), one CS1-CS3-positive E. coli strain producing ST only was found. As described previously (8, 31), the antigens CS1 and CS2 were only associated with the serotypes 06:K15:H16 and H-, whereas CS3 was associated with other serotypes (8, 27). In this study, the CS1-CS3-positive strains were found to belong to 06:K15:H16, whereas the CS2-CS3-positive strain was H-. Two reports (38, 43) have shown that CFA/IV-positive strains hemagglutinate human, bovine, and/or guinea pig erythrocytes. In this study we found that strains producing CS5 and CS6 rarely gave this HA pattern; instead, most of them agglutinated sheep erythrocytes. These results may suggest differences in the binding structures on the erythrocytes within the same animal species (8, 12). All of the serotype 0128:H21 strains expressed CS5 and CS6. No strains produced CS4 and CS6, and none of the serotypes reported to be associated with these adhesins (27, 39) were found. Knutton et al. (18) have suggested that the presence of CS6 only would not be enough to enable bacteria to attach to enterocytes. Their ultrastructural studies have shown the presence of other fimbrial or fibrillar surface antigens that probably represent surface antigens responsible for the adhesive properties in ETEC of 0148:H28, 0159:H20, 027: H7, and 027:H20 serotypes. Since two of the three strains with CS6 only in this study belonged to the serotype 0148: H28, it is possible that the putative colonization factor PCF0148 (17) may be present on these strains. Studies in a rabbit nonligated intestine model, on the other hand, have shown that ETEC strains expressing CS6 alone colonize rabbit intestine significantly better than do corresponding CS-deficient mutant strains (33). The existence of more than one of the coinactivated CFAs, i.e., CFA/I, CFA/II, and CFA/IV, in the same ETEC isolate has never been reported. Furthermore, in spite of an intense search in more than 400 natural ETEC isolates for strains that may express, e.g., CFA/I together with CFA/II or CFA/IV or CFA/II together with CFA/IV, we have failed to identify such strains (unpublished data). However, studies are in progress to construct suitable live vaccine strains and to introduce several of the CFAs in the same host organism by recombinant DNA

techniques. The mechanisms by which cells can bind to each other are many and varied. They include lectinlike interactions, electrostatic and hydrophobic interactions, hydrogen bonds, and Van der Waals interactions (21). Hydrophobic interaction has been thought to be of major importance in the attachment of the CFA/I- and CFA/II-positive strains to host cells; therefore methods determining hydrophobic binding may be used for indirect measurements of CFA properties. However, all of the CFA/IV-positive strains tested did not show

VOL. 29, 1991

ETEC COLONIZATION FACTORS IN ARGENTINA

hydrophobic properties with the SO test. It may be inferred that other mechanisms of attachment to enterocytes exist in CFA/IV-positive strains. Among the 52 strains for which no colonization factor could be identified, 50% produced ST, 44% produced LT, and 6% produced both. Of the 26 ST-positive ETEC strains, 17 (65%) showed MRHA with some of the erythrocytes tested and/or hydrophobicity, whereas only 35% of the LT-positive ETEC strains exhibited either of these properties. This may suggest a high frequency of adhesins not associated with either hemagglutination or hydrophobicity, particularly in strains producing LT only. Since ETEC bacteria probably have to colonize the small intestine by some adherence factor to cause disease, the relatively high frequency of nonhemagglutinating, nonhydrophobic strains in this study suggests that additional methods would be required to screen for other colonization factors. Recently a number of different putative colonization factor antigens have been described, e.g., PCF09, PCF0166, and CS17 (14, 25, 26), which have been associated with the serotypes 09:H-, 078:H18, and 0114:H21, respectively. These serotypes and several others that may be associated with putative new colonization factors were observed among the 52 CFA-negative strains (data not shown). Thus, e.g., one 078:H18 ST-positive ETEC strain hemagglutinated human and bovine erythrocytes only when it had been grown in the presence of bile salts, suggesting the expression of PCF0166 (25). Likewise, an 09:H- LT-positive ETEC strain showing the same characteristic as the PCF09 strain previously reported (14) was found. Furthermore, two defined clusters of strains with a characteristic HA pattern can be clearly seen, suggesting the presence of at least two probable new colonization factors. In a subsequent study we will try to identify some of the new putative colonization factors in these 52 strains to increase our knowledge of the prevalence of these CFAs on ETEC strains causing diarrhea in Argentina. It has been suggested that an effective ETEC vaccine should contain the most prevalent colonization factor antigens (32). Therefore, it is important to know the prevalence of the different CFAs on ETEC strains in various geographic areas and to identify additional colonization factors for the CFA-negative ETEC strains.

4. Black, R. E. 1986. The epidemiology of cholera and enterotoxigenic Escherichia coli diarrheal disease, p. 23-32. In J. Holmgren, A. Lindberg, and R. Moilby (ed.), Development of vaccine and drugs against diarrhea. 11th Nobel Conference, Stockholm, Studentlitteratur, Lund, Sweden. 5. Blanco, J., E. A. Gonzalez, M. Blanco, J. L. Garabal, P. Alonso, W. H. Jansen, and P. A. M. Guinee. 1989. Prevalence of enterotoxigenic Escherichia coli strains in outbreaks and sporadic cases of diarrhea in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 8:396-400. 6. Changchawalit, S., P. Echeverria, D. N. Taylor, U. Leksomboon, C. Tirapat, B. Eampokalap, and B. Rowe. 1984. Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand. Infect. Immun. 45:525-527. 7. Cravioto, A., R. E. Reyes, R. Ortega, G. Ferniindez, R. Hernandez, and D. L6pez. 1988. Prospective study of diarrhoeal disease in a cohort of rural Mexican children: incidence and isolated pathogens during the first two years of life. Epidemiol. Infect. 101:123-134. 8. Cravioto, A., S. M. Scotland, and B. Rowe. 1982. Hemagglutination activity and colonization factor antigens I and II in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli isolated from humans. Infect. Immun. 36:189-197. 9. Evans, D. G., and D. J. Evans, Jr. 1978. New surface associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli of serogroups 06 and 08. Infect. Immun. 21:638-647. 10. Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G. Chase, and S. L. Gorbach. 1975. Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect. Immun. 12:656-667. 11. Evans, D. J., Jr., D. G. Evans, and H. L. DuPont. 1979. Hemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with human, bovine, chicken, and guinea pig erythrocytes in the presence and absence of mannose. Infect. Immun. 23:336-346. 12. Faris, A., J. Sellei, M. Lindahl, and T. Wadstrom. 1982. Hemagglutination of bovine erythrocytes by enterotoxigenic Escherichia coli (ETEC) of 06 serogroup: evidence for glycoconjugate receptor heterogeneity. Zentralbl. Bakteriol. Parasiterkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 253:175-182. 13. Gothefors, L., C. Ahren, B. Stoll, D. K. Barua, F. 0rskov, M. A. Salek, and A. M. Svennerholm. 1985. Presence of colonization factor antigens on fresh isolates of fecal Escherichia coli: a prospective study. J. Infect. Dis. 152:1128-1133. 14. Heuzenroeder, M. W., T. R. Elliot, C. J. Thomas, R. Halter, and P. A. Manning. 1990. A new fimbrial type (PCFO9) on enterotoxigenic Escherichia coli 09:H- LT' isolated from a case of infant diarrhea in Central Australia. FEMS Microbiol. Lett.

ACKNOWLEDGMENTS We thank R. Notario, A. M. Picandet, M. Vergara, E. Patrito, 0. Nader, D. Maurel, and M. Szefner for supplying the E. coli strains. We gratefully acknowledge the technical assistance of Gustavo Basanta, Marcelo Lifschitz, Ana Garbini, Kerstin Andersson, and Gudrun Wiklund. Financial support was obtained from the Swedish Agency for Research Cooperation with Developing Countries, the Secreteriat of Science and Technology, Argentina, and the Swedish Medical Research Council (grant 16X-09084).

REFERENCES 1. Aguero, M. E., L. Reyes, V. Prado, I. Orskov, F. Orskov, and F. C. Cabello. 1985. Enterotoxigenic Escherichia coli in a

population of infant with diarrhea in Chile. J. Clin. Microbiol. 22:576-581. 2. Ahren, C. M., and A.-M. Svennerholm. 1982. Synergistic protective effect of antibodies against Escherichia coli enterotoxin and colonization factor antigens. Infect. Immun. 38:74-79. 3. Ahren, C. M., and A.-M. Svennerholm. 1985. Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type. Infect. Immun. 50:255-261.

1897

66:55-60. 15. Honda, T., M. Arita, and T. Miwatani. 1984. Characterization of new hydrophobic pili of human enterotoxigenic Escherichia coli: a possible new colonization factor. Infect. Immun. 43:959965. 16. Honda, T., M. M. Khan, Y. Takeda, and T. Miwatani. 1983. Grouping of enterotoxigenic Escherichia coli by hydrophobicity and its relation to hemagglutination and enterotoxin production. FEMS Microbiol. Lett. 17:273-276. 17. Knutton, S., D. R. Lloyd, and A. S. McNeish. 1987. Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype 0148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor. Infect. Immun. 55:86-92. 18. Knutton, S., M. M. McConnell, B. Rowe, and A. S. McNeish. 1989. Adhesion and ultraestructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV. Infect. Immun. 57:3364-3371. 19. Levine, M. M., R. E. Black, H. L. Clements, C. R. Young, C. P. Cheney, P. Schad, H. Collins, and E. C. Boedeker. 1984. Prevention of enterotoxigenic Escherichia coli diarrheal infection in man by vaccines that stimulate anti-adhesion (anti-pili) immunity, p. 223-224. In E. C. Boedeker (ed.), Attachment of microorganisms to the gastrointestinal mucosal surface. CRC

1898

BINSZTEIN ET AL.

Press, Boca Raton, Fla. 20. Lindahl, M., A. Faris, T. Wadstrom, and S. Hjerten. 1981. A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells. Biochem. Biophys. Acta 677: 471-476. 21. Lopez-Vidal, Y., J. J. Calva, A. Trujillo, A. Ponce de Leon, A. Ramos, A.-M. Svennerholm, and B. R. Ruiz Palacios. 1990. Enterotoxins and adhesins of enterotoxigenic Escherichia coli: are they risk factors for acute diarrhea in the community? J. Infect. Dis. 162:442-447. 22. Lopez-Vidal, Y., P. Klemm, and A.-M. Svennerholm. 1988. Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin producing Escherichia coli. J. Clin. Microbiol. 26:1967-1972. 23. Lopez-Vidal, Y., and A.-M. Svennerholm. 1986. Hemadsorption and enzyme-linked immunosorbent assay nitrocellulose replica methods for identification of colonization factor antigen (CFA)positive Escherichia coli colonies and for isolation of CFAnegative mutants. J. Clin. Microbiol. 24:615-619. 24. Lopez-Vidal, Y., and A.-M. Svennerholm. 1990. Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 28:1906-1912. 25. McConnell, M. M., H. Chart, A. M. Field, M. Hibberd, and B. Rowe. 1989. Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup 0166. J. Gen. Microbiol. 135:1135-1144. 26. McConnell, M. M., M. Hibberd, A. M. Field, H. Chart, and B. Rowe. 1990. Characterization of a new colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype 0114:H21 which produces only heat-labile enterotoxin. J. Infect. Dis. 161:343-347. 27. McConnell, M. M., L. V. Thomas, N. P. Day, and B. Rowe. 1985. Enzyme-linked immunosorbent assays for the detection of adhesion factor antigens of enterotoxigenic Escherichia coli. J. Infect. Dis. 152:1120-1127. 28. Ouchterlony, O., and L. A. Nilsson. 1978. Immunodiffusion and immunoelectrophoresis, p. 1-44. In D. M. Weir (ed.), Handbook of experimental immunology, 3rd. ed. Blackwell Scientific Publications, Ltd., Oxford. 29. Reis, M. H. L., B. E. C. Guth, T. A. T. Gomes, J. Murahovsk, and L. R. Trabulsi. 1982. Frequency of Escherichia coli strains producing heat-labile toxin or heat-stable toxin or both in children with and without diarrhea in Sao Paulo. J. Clin. Microbiol. 15:1062-1064. 30. Smith, H. R., S. M. Scotland, and B. Rowe. 1983. Plasmids that code for production of colonization factor antigen II and enterotoxin production in strains of Escherichia coli. Infect. Immun. 40:1236-1239. 31. Smyth, C. J. 1982. Two mannose-resistant haemagglutinins on enterotoxigenic Escherichia coli of serotype 06:K15:H16 or Hisolated from travellers and infantile diarrhea. J. Gen. Microbiol. 128:2081-2096.

J. CLIN. MICROBIOL. 32. Svennerholm, A.-M., J. Holmgren, and D. A. Sack. 1989. Development of oral vaccines against enterotoxigenic Escherichia coli diarrhea. Vaccine 7:196-198. 33. Svennerholm, A.-M., Y. Lopez-Vidal, J. Holmgren, M. M. McConnell, and B. Rowe. 1988. Role of PCF8775 antigen and its coli surface subcomponents for colonization, disease, and prospective immunogenicity of enterotoxigenic Escherichia coli in rabbits. Infect. Immun. 56:523-528. 34. Svennerholm, A.-M., and G. Wiklund. 1983. Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17:596-600. 35. Svennerholm, A.-M., M. Wikstrom, M. Lindblad, and J. Holmgren. 1986. Monoclonal antibodies against Escherichia coli heat-stable toxin (ST) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay. J. Clin. Microbiol. 24:585-590. 36. Tacket, C. O., D. R. Maneval, and M. M. Levine. 1987. Purification, morphology and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli 0159: H4. Infect. Immun. 55:1063-1069. 37. Thomas, L. V., A. Cravioto, S. M. Scotland, and B. Rowe. 1982. New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans. Infect. Immun. 35:1119-1124. 38. Thomas, L. V., M. M. McConnell, B. Rowe, and A. M. Field. 1985. The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775. J. Gen. Microbiol. 131:2319-2326. 39. Thomas, L. V., and B. Rowe. 1982. The occurrence of colonization factors (CFA/I, CFA/II and E8775) in enterotoxigenic Escherichia coli from various countries in South East Asia. Med. Microbiol. Immunol. 171:85-90. 40. Wadsworth, C. 1959. A microplate technique employing a gel chamber compared with other micro and macroplate technique for immunodiffusion. Int. Arch. Allergy Appl. Immunol. 21: 131-137. 41. Willshaw, G. A., M. M. McConnell, H. R. Smith, and B. Rowe. 1990. Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025:H42. FEMS Mi2crobiol. Lett. 68:255-260. 42. Willshaw, G. A., H. R. Smith, M. M. McConnell, E. A. Barclay, J. Krnjulac, and B. Rowe. 1982. Genetic and molecular studies of plasmids coding for colonization factor antigen I and heatstable enterotoxin in several Escherichia coli serotypes. Infect. Immun. 37:858-868. 43. Wolf, M. K., G. P. Andrews, B. D. Tall, M. M. McConnell, M. M. Levine, and E. C. Boedeker. 1989. Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775. Infect. Immun. 57:164-173.