Vol. 28, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1990, p. 2264-2268
0095-1137/90/102264-05$02.00/0 Copyright © 1990, American Society for Microbiology
Detection of Colonization Factor Antigen I-Positive Enterotoxigenic Escherichia coli with a Cloned Polynucleotide Probe HARLEEN M. S. GREWAL,12t* HALVOR SOMMERFELT,1 WIM GAASTRA,3 ANN-MARI SVENNERHOLM,4 MAHARAJ K. BHAN,5 ANJA M. HAMERS,3 RAMESH KUMAR,6 GUDRUN WIKLUND,4 ANDBJARNE BJORVATN1
Institute of International Health and Medical Department B, Haukeland Hospital,' and Center of Biotechnology/ University of Bergen, 5021 Bergen, Norway; Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands3; Department of Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden4; and Department of Pediatrics, Division of Gastroenterology and Enteric Infections,s and Department of Microbiology,6 All India Institute of Medical Sciences, New Delhi, India Received 27 February 1990/Accepted 11 July 1990
We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.
Enterotoxigenic Escherichia coli (ETEC) is a frequent of diarrheal disease among humans and young animals (31, 37). Colonization of the small intestine and delivery of the heat-stable toxin (ST), the heat-labile toxin (LT), or both in close proximity to the enterocytes are prerequisites for the development of disease (19, 37). ETEC strains possess fimbriae or pili, which serve as colonization factors and which mediate attachment to receptors on the intestinal mucosa (19, 27). In strains pathogenic to humans, a number of colonization factor antigens (CFAs) have been identified, including CFA/I, CFA/Il, CFA/IV (PCF8775), CFA/III, PCF0166, and PCFO159 (7, 10, 17, 20, 22, 25, 39, 40). CFA/I has a uniform fimbrial structure, while CFA/Il is composed of three distinct components designated E. coli surfaceassociated antigens CS1, CS2, and CS3, which are present in different permutations. Similarly, CFA/IV comprises CS4, CS5, and CS6 (6, 32, 40). The genes required for the expression and assembly of CFA/I have been found to be located on plasmids that also encode ST (10, 29, 30). The structural CFA/I gene has been subcloned and its nucleotide sequence has been deciphered (15, 18). In the CFA/I-ST plasmid NTP113, two regions separated by approximately 40 kilobase pairs are required for the production of CFA/I fimbriae (28, 30). Region 1 directs the production of at least five polypeptides, one of which is the fimbrial subunit (15; W. Gaastra, unpublished data), while region 2 codes for a polypeptide of 265 amino acid residues, which acts as a positive regulator for the expression of CFA/I genes by binding to the promoter site of the CFA/I region 1 DNA (28). The nucleotide sequences encoding CS1 and CS2 are not known, but the N-terminal
amino acid sequences may share some degree of homology with those of CFA/I and CS4 (3, 19, 42). Antibodies against CFA/I are important in preventing mucosal colonization by CFA/I-bearing strains (9, 37). Immunization with purified CFA/I has been reported to induce protective immunity in humans (9). Vaccines based on antigens derived from enterotoxins and CFAs are being developed against ETEC infections (37). Such vaccines should contain CFAs of the ETEC strains that prevail in target populations. The prevalence of the various CFAs associated with human ETEC strains shows considerable geographic variation (4, 14, 37, 41). Thus, accurate and simple techniques for the identification of CFAs are indispensable for the development of vaccines against ETEC. Surveys have been carried out by using mannose-resistant hemagglutination (6, 14) and immunodiffusion (14) tests. The mannose-resistant hemagglutination tests are not very specific however; and the immunodiffusion tests are cumbersome and have suboptimal sensitivities (1, 24). Enzymelinked immunosorbent assays (ELISAs) (21, 24) are simple to perform and have a high level of sensitivity and specificity. However, the antibodies required for ELISA are not readily available. The host range of ETEC is determined by the specific interaction of a particular adhesin produced by the bacterium and a receptor present on the intestinal epithelial cells of the host (11, 13). Thus, the colonization factors of human ETEC strains are quite different from the fimbriae K88 and 987P found on porcine; F41 on bovine; and K99 on calf, lamb, and piglet pathogens (13). In studies of environmental sources of human diarrheal pathogens, detection of ETEC toxin genes by using the available gene probe assays (16, 26, 33, 36) lacks specificity, as even animal strains test positive with the LT and one of the ST probes (36). Thus, development of a detection technique specific for human ETEC is
cause
Corresponding author. t Present address: Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. *
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TABLE 1. Characteristics of human ETEC strains used for calibration and evaluation of the CFA/I hybridization assay No. of strains
1 8 1 4 2 3 3 4 1 1 1 1 30 30 45
Enterotoxin profile ST
LT
+ + + + + + + + +
+ +
+ + + +
-
+ + + +
+ + +
+
+
Type of fimbriae
QOrigina
CFA/I+ CFA/I+ CFA/I+ CS1+ CS3+ CS2+ CS3+ CS3+ CS4+ CS6+ CS5+ CS6+ CS5+ CS6+ CS5+ CS6+ CS4+ CS6+ CS5+ CS6+ Uncharacterized Uncharacterized Uncharacterized
A B B C C C C D
C D E E F F F
a Origins: A, strain H10407+ (provided by D. G. Evans, Houston, Tex.); B, Dhaka, Bangladesh (14); C, Dhaka, Bangladesh (5); D, Argentina (provided by N. Binzstein, Institute Malbran, Buenos Aires); E, Provided by B. Rowe (Colindale, London, England); F, India (2, 35).
essential for further epidemiological studies of ETEC transmission. We describe here a colony hybridization assay in which we used a cloned fragment of the CFA/I region 1 DNA containing the promoter and part of the cfaA gene (15). The CFA/I gene probe assay was evaluated against an established CFA/I monoclonal antibody (MAb)-based inhibition ELISA.
MATERIALS AND METHODS Bacterial strains. The characteristics of the strains used for calibrating and evaluating the hybridization assay are given in Table 1. The toxin profiles were determined by using an established oligonucleotide colony hybridization assay (36). Strains with previously determined CFA profiles were used for initial calibration of the washing conditions of the hybridization assays. CFA/I was identified previously by a MAbbased ELISA (21); and CS1, CS2, and CS3 (21a) and CS4 and CS5 (unpublished data) were identified by both ELISA and slide agglutination tests. CS6 was identified previously by using polyclonal antisera in a double-diffusion assay (38). In addition, for evaluating the CFA/I probe, 105 previously uncharacterized ETEC isolates obtained from Indian children with acute and persistent diarrhea (2, 35) were studied. Thus, a total of 135 human ETEC isolates were used to test the characteristics of the CFA/I hybridization assay. In addition, a strain harboring pBS (Stratagene, La Jolla, Calif.), the plasmid vector of the CFA/I probe, was examined to exclude the possibility of probe contamination by vector DNA. For further evaluation of the specificity of the probe, we included eight ETEC strains isolated from animals with diarrhea as well as nine non-ETEC isolates from healthy children. Cultivation of bacteria and inoculation of nitrocellulose membranes. All strains were grown overnight on MacConkey agar at 37°C. Three clones from each strain were examined in parallel by the CFA/I inhibition ELISA and by colony hybridization. To avoid discrepancies caused by the spontaneous loss of CFA/I genes (34), the same three clones from each bacterial isolate were inoculated on sepa-
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rate CFA agar plates (8) and onto nitrocellulose membranes
(Schleicher & Schuell, Inc., Keene, N.H.) overlying fresh MacConkey agar, following the guidelines described earlier (36). The CFA agar plates were incubated overnight at 37°C, while the nitrocellulose membranes were processed as described previously (36). Probe preparation and labeling. pIVB3-c17, a pBS derivative containing a DNA fragment between the first PstI and EcoRI sites of CFA/I region 1 DNA (15), was cleaved with PstI and EcoRI, and the resulting fragments were electrophoretically separated on a 1% agarose gel. The larger of the two fragments, approximately 1,000 base pairs, was isolated for use as the CFA/I probe with the Geneclean kit (Bio 101 Inc., La Jolla, Calif.) according to the instructions of the manufacturer. Similarly, to detect the presence of the regulatory gene, the EcoRI-XbaI fragment of CFA/I region 2, which contains the gene encoding the regulatory protein (28), was used as a probe, termed cfaD. Fifty nanograms of each probe was radiolabeled with 32P as described by Feinberg and Vogelstein (12). A specific activity of 1.5 x 109 cpm/,ug was achieved. Colony hybridization and calibration of washing conditions. Colony hybridization was performed as described earlier (35). Initially, the 30 strains with known CFA profiles were used to determine the optimal washing conditions (33). Thus, after radiolabeling and hybridization, the salt concentration was decreased stepwise from 3x to 0.05x SSC (lx SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate) by using a constant temperature of 67°C. CFA/I inhibition ELISA. The capacity of CFA/I-carrying bacteria to inhibit the binding of anti-CFA/I MAbs to solidphase-bound CFA/I was assayed by a previously described ELISA (21). In brief, ELISA microdilution plates (Nunc, A/S Roskilde, Denmark) were coated with 0.5 p.g of purified CFA/I per ml, 100 ,ul per well, at 37°C overnight. After the plates were washed in phosphate-buffered saline (PBS; pH 7.2), they were blocked with 0.1% bovine serum albumin (BSA) in PBS (PBS-BSA) at 37°C for 30 min. Bacterial suspensions were made by dissolving a loopful of bacteria cultured on CFA agar in 100 ,ul of PBS-BSA. Sixty microliters of each bacterial suspension was added to individual, blocked wells. Anti-CFA/I MAb 1:6 (21) diluted 1/100 in PBS-BSA was added (60 pl per well), and the plates were incubated for 90 min at room temperature. After washing in PBS with 0.05% Tween 20 (PBS-Tween), 100 pul of antimouse immunoglobulin-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories Inc.) diluted 1/2,000 in PBS-Tween-BSA was added per well. After incubation for an additional 90 min, the plates were developed with an o-phenylenediamine-H202 enzyme substrate. Interpreting the test results of CFA/I colony hybridization and ELISA. To minimize bias, the tests were read independently by two investigators before the reference code was broken. Bacterial suspensions that caused 250% inhibition in the ELISA were considered positive. The results were read both visually and spectrophotometrically. The ELISA and hybridization results were interpreted by predefined criteria (36). Thus, a strain was considered CFA/I negative if two or three colonies showed adequate growth and the test results for these were negative. If at least one of the colonies gave a positive result, the strain was considered CFA/I positive regardless of growth pattern. If two of the colonies did not show adequate growth and the third colony gave a negative test result, both assays would be repeated. Examination of strains testing positive in the CFA/I hybridization assay. All strains that tested positive in the CFA/I
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TABLE 2. Parallel examination of 135 ETEC strains by CFA/I inhibition ELISA and CFA/I gene probe hybridization Phenotype by ELISA
CFA/I+ CFA/I-
FIG. 1. Autoradiograms of two nitrocellulose membranes containing DNA from the same three ETEC strains after hybridization with a 32P-labeled CFA/I gene probe. The vertical sequence of inoculation starts from the top of the far left column. Five control strains of E. coli (H10407+ harboring the CFA/I gene, a strain with the probe vector, a non-ETEC strain, a CS5+ CS6+ strain, and a CS4+ CS6+ strain) were inoculated onto column 1. Three test strains (18 colonies from each) were examined in columns 2 to 4, 5 to 7, and 8 to 10, respectively. After hybridization, stringent washing of both membranes was performed at 67°C but with different ionic concentrations. The autoradiograms in panels A and B were obtained after washing in lx SSC-0.1% SDS and 0.07x SSC-0.1% SDS, respectively. Strains 1 and 3 (columns 2 to 4 and 8 to 10) yielded clones that produced CFA/I, while clones from strain 2 (columns 5 to 7) were previously shown to produce CS4. The CS4 gene was only detected after low-stringency washing (A), whereas the CFA/I gene was also identified after high-stringency washing (B). Probe-negative clones in columns 3, 4, 7, and 9 probably lost their CFA genes spontaneously. These clones did not produce CFA/I or CS4.
hybridization assay were further examined with the cfaD probe. Strains that showed discrepant results between CFA/I hybridization and ELISA were examined further. Thus, such isolates were again cultured from the original stab agars. Revived clones were tested along with the initially examined clones by slide agglutination using antiCFA/I MAb 1:6 (21). From each isolate, two agglutinating and four nonagglutinating clones were examined by both genotypic assays. RESULTS Standardization of washing conditions. After stringent washing of the hybridized CFA/I gene probe in a solution of lx SSC-0.1% sodium dodecyl sulfate (SDS), some of the colony lysates yielded signals which gradually disappeared when the stringency was increased. These signals disappeared completely after the probe was washed in 0.07x SSC-0.1% SDS, while other signals maintained their intensities (Fig. 1). The strains that were positive on low-stringency washing were previously shown to produce either CFA/I or CS4 (Table 1). After washing at high stringency, only CFA/I strains were detected (Fig. 1). Thus, for evaluating the efficiency of the CFA/I probe, the stringent washes were performed in 0.07x SSC-0.1% SDS at 67°C. Similar calibration of the cfaD probe assay indicated an optimum signal/background ratio after the probe was washed with 0.lx SSC-0.1% SDS at 67°C. No hybridization was observed between the CFA/I probe and DNA from the CS1+ CS3+-, CS2+ CS3+-, CS3+-, or CS5+ CS6+-producing strains, even after low-stringency washing. The eight ETEC strains of animal origin, the 9 non-ETEC strains, and the strain harboring the plasmid vector of the polynucleotide gene probe were all negative in the CFA/I hybridization assay.
No. with the following genotype by hybridization:
CFA/I+
CFA/I-
23 1
111
0
Detection of CFA/I-bearing ETEC by CFA/I ELISA and colony hybridization. Analyses of the 135 human ETEC isolates showed a high level of concordance between the CFA/I ELISA (both visually and spectrophotometrically) and the CFA/I hybridization assay (Table 2). Of the 10 previously characterized CFA/I strains, 8 tested positive in the ELISA as well as in the hybridization assay. However, strain 251575-2 was negative in both assays. Furthermore, all three clones of strain 254175-2, while negative in the ELISA, yielded positive signals in the hybridization assay. Similarly, one of three clones of the Indian strain Bh3-10 showed inconsistency in the gene probe assay and the ELISA. Thus, whereas the DNAs from all three clones hybridized with the CFA/I probe, only two of the clones produced CFA/I, as determined by ELISA. This strain was, by our predefined criteria, typed as CFA/I positive in both assays. Of the remaining 104 Indian ETEC strains, 14 were concomitantly positive in the genotypic and phenotypic CFA/I assays. Thus, altogether, 23 strains tested positive in the CFA/I ELISA, whereas 24 tested positive in the CFA/I hybridization assay. Of the 72 (24 x 3) CFA/I probe-positive clones, only the 4 (3 from strain 254175-2 and 1 from strain Bh3-10) that did not produce CFA/I were negative in the cfaD hybridization assay. Additional clones of these two strains were revived and examined by CFA/I slide agglutination. The two agglutinating clones (from strain 254175-2) were positive in both hybridization assays. On the other hand, one of the four nonagglutinating clones tested positive in the CFA/I hybridization assay, whereas it was negative in the cfaD hybridization assay. The remaining three CFA/I- clones were negative in both gene probe assays. Identical results were obtained on examination of strain Bh3-10. Strain 251575-2, which was previously classified as ST+ LT+ CFA/I+ (14), was found to be negative in both hybridization assays and in the CFA/I ELISA. Repeat examination by the ST probe assay showed that this strain lost its ST gene. In contrast, the four clones (three from strain 254175-2 and one from strain Bh3-10) that were positive in the CFA/I hybridization assay but negative in the cfaD hybridization assay and the CFA/I ELISA maintained their ST genes.
DISCUSSION Parallel examination of 135 ETEC strains with the CFA/I probe and in the CFA/I ELISA showed a high level of correspondence in the identification of strains expressing CFA/I. However, all three clones of one strain were positive in the CFA/I hybridization assay but negative in the ELISA. Interestingly, this strain was previously shown to produce CFA/I (14). A similar discrepant result was obtained in one of three clones of another strain, with the remaining two clones being correspondingly positive in the genotypic and phenotypic tests for CFA/I. Further examination of these four clones (from two strains) that lacked CFA/I revealed the loss of a regulatory gene component. In contrast, all CFA/I
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producers, including additional clones revived from the same two strains, had an intact regulatory gene. Hence, the discrepant results between the CFA/I hybridization assay and the ELISA may reflect an artificially induced inactivation of the previously active CFA/I gene because of longterm storage and repeated subcultures. This observation suggests that studies of CFAs, especially with ETEC strains stored for long periods, may benefit from the detection ofthe CFA genes directly rather than the detection of the gene products. The finding of a concomitant loss of the ST and the CFA/I genes in one strain corroborates the results of earlier reports on the close link between these two genes (10, 29, 30, 34). Interestingly, after low-stringency washing, the CFA/I probe also detected ETEC strains that were previously shown to produce CS4 (H. Sommerfelt et al., unpublished data). This indicates a considerable nucleotide sequence homology between the 5' end of the CFA/I region 1 DNA and CS4-associated nucleotide sequences, thereby implying a close genetic relationship between these two fimbrial structures. This interpretation is supported by the amino acid sequence homology observed between the N-terminal ends of CFA/I and CS4 (42) and the strong immunological cross-reactivity between these two fimbrial antigens (23). In epidemiological studies on the transmission of enteric pathogens, the CFA/I probe evaluated in this study and future probes for other host-specific colonization factors offer a distinct advantage in distinguishing among human and animal ETEC strains. Nonradioactive labeling (33) of such probes will further facilitate characterization of ETEC strains. The application of such probes in epidemiological studies should provide the information needed for future construction of vaccines against diarrhea caused by ETEC strains. ACKNOWLEDGMENTS We thank Curt Endressen and Dag Helland, Center of Biotechnology, as well as Claus Ola Solberg, Medical Department B, University of Bergen, for providing excellent laboratory facilities. The skillful technical assistance of Vibecke Asphaug is gratefully appreciated. The financial support of the Norwegian Research Council for Science and the Humanities, the Norwegian Ministry of Development Cooperation, and the Swedish Medical Research Council is gratefully appreciated. LITERATURE CITED 1. Âhrén, C. M., L. Gothefors, B. J. Stoll, M. A. Salek, and A.-M. Svennerholm. 1986. Comparison of methods for detection of colonization factor antigens on enterotoxigenic Escherichia
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probe assay for identification of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 28:49-54. Sommerfelt, H., B.-I. Haukanes, K. H. Kalland, A.-M. Svennerholm, J. Sanchéz, and B. Bjorvatn. 1989. Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli. APMIS 97:436-440. Sommerfelt, H., K. H. Kalland, P. Raj, S. L. Moseley, M. K. Bhan, and B. Bjorvatn. 1988. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 26:2275-2278. Sommerfelt, H., A.-M. Svennerholm, K. H. Kalland, B.-I. Haukanes, and B. Bjorvatn. 1988. Comparative study of colony hybridization using synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for the identification of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 26:530-534. Svennerholm, A.-M., J. Holmgren, and D. A. Sack. 1989. Development of oral vaccines against enterotoxigenic Escherichia coli diarrhoea. Vaccine 7:196-198. Svennerholm, A.-M., Y. Lopéz-Vidal, J. Holmgren, M. M. McConell, and B. Rowe. 1988. Role of PCF8775 antigen and its coli surface subcomponents for colonization, disease, and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits. Infect. Immun. 56:523-528. Tacket, C. O., D. R. Maneval, and M. M. Levine. 1987. Purification, morphology, and genetics of a new fimbrial putative colonization factor in enterotoxigenic Escherichia coli 0159:H4. Infect. Immun. 55:1063-1069. 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. Thomas, L. V., and B. Rowe. 1982. The occurrence of colonization factors (CFA/I, CFA/Il and E8775) in enterotoxigenic Escherichia coli from various countries in South East Asia. Med. Microbiol. Immunol. 171:85-90. Wolf, M. K., G. P. Andrews, B. D. Tall, M. M. McConnell, and E. C. Boedecker. 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.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1990, p. 2264-2268
0095-1137/90/102264-05$02.00/0 Copyright © 1990, American Society for Microbiology
Detection of Colonization Factor Antigen I-Positive Enterotoxigenic Escherichia coli with a Cloned Polynucleotide Probe HARLEEN M. S. GREWAL,12t* HALVOR SOMMERFELT,1 WIM GAASTRA,3 ANN-MARI SVENNERHOLM,4 MAHARAJ K. BHAN,5 ANJA M. HAMERS,3 RAMESH KUMAR,6 GUDRUN WIKLUND,4 ANDBJARNE BJORVATN1
Institute of International Health and Medical Department B, Haukeland Hospital,' and Center of Biotechnology/ University of Bergen, 5021 Bergen, Norway; Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands3; Department of Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden4; and Department of Pediatrics, Division of Gastroenterology and Enteric Infections,s and Department of Microbiology,6 All India Institute of Medical Sciences, New Delhi, India Received 27 February 1990/Accepted 11 July 1990
We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.
Enterotoxigenic Escherichia coli (ETEC) is a frequent of diarrheal disease among humans and young animals (31, 37). Colonization of the small intestine and delivery of the heat-stable toxin (ST), the heat-labile toxin (LT), or both in close proximity to the enterocytes are prerequisites for the development of disease (19, 37). ETEC strains possess fimbriae or pili, which serve as colonization factors and which mediate attachment to receptors on the intestinal mucosa (19, 27). In strains pathogenic to humans, a number of colonization factor antigens (CFAs) have been identified, including CFA/I, CFA/Il, CFA/IV (PCF8775), CFA/III, PCF0166, and PCFO159 (7, 10, 17, 20, 22, 25, 39, 40). CFA/I has a uniform fimbrial structure, while CFA/Il is composed of three distinct components designated E. coli surfaceassociated antigens CS1, CS2, and CS3, which are present in different permutations. Similarly, CFA/IV comprises CS4, CS5, and CS6 (6, 32, 40). The genes required for the expression and assembly of CFA/I have been found to be located on plasmids that also encode ST (10, 29, 30). The structural CFA/I gene has been subcloned and its nucleotide sequence has been deciphered (15, 18). In the CFA/I-ST plasmid NTP113, two regions separated by approximately 40 kilobase pairs are required for the production of CFA/I fimbriae (28, 30). Region 1 directs the production of at least five polypeptides, one of which is the fimbrial subunit (15; W. Gaastra, unpublished data), while region 2 codes for a polypeptide of 265 amino acid residues, which acts as a positive regulator for the expression of CFA/I genes by binding to the promoter site of the CFA/I region 1 DNA (28). The nucleotide sequences encoding CS1 and CS2 are not known, but the N-terminal
amino acid sequences may share some degree of homology with those of CFA/I and CS4 (3, 19, 42). Antibodies against CFA/I are important in preventing mucosal colonization by CFA/I-bearing strains (9, 37). Immunization with purified CFA/I has been reported to induce protective immunity in humans (9). Vaccines based on antigens derived from enterotoxins and CFAs are being developed against ETEC infections (37). Such vaccines should contain CFAs of the ETEC strains that prevail in target populations. The prevalence of the various CFAs associated with human ETEC strains shows considerable geographic variation (4, 14, 37, 41). Thus, accurate and simple techniques for the identification of CFAs are indispensable for the development of vaccines against ETEC. Surveys have been carried out by using mannose-resistant hemagglutination (6, 14) and immunodiffusion (14) tests. The mannose-resistant hemagglutination tests are not very specific however; and the immunodiffusion tests are cumbersome and have suboptimal sensitivities (1, 24). Enzymelinked immunosorbent assays (ELISAs) (21, 24) are simple to perform and have a high level of sensitivity and specificity. However, the antibodies required for ELISA are not readily available. The host range of ETEC is determined by the specific interaction of a particular adhesin produced by the bacterium and a receptor present on the intestinal epithelial cells of the host (11, 13). Thus, the colonization factors of human ETEC strains are quite different from the fimbriae K88 and 987P found on porcine; F41 on bovine; and K99 on calf, lamb, and piglet pathogens (13). In studies of environmental sources of human diarrheal pathogens, detection of ETEC toxin genes by using the available gene probe assays (16, 26, 33, 36) lacks specificity, as even animal strains test positive with the LT and one of the ST probes (36). Thus, development of a detection technique specific for human ETEC is
cause
Corresponding author. t Present address: Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. *
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TABLE 1. Characteristics of human ETEC strains used for calibration and evaluation of the CFA/I hybridization assay No. of strains
1 8 1 4 2 3 3 4 1 1 1 1 30 30 45
Enterotoxin profile ST
LT
+ + + + + + + + +
+ +
+ + + +
-
+ + + +
+ + +
+
+
Type of fimbriae
QOrigina
CFA/I+ CFA/I+ CFA/I+ CS1+ CS3+ CS2+ CS3+ CS3+ CS4+ CS6+ CS5+ CS6+ CS5+ CS6+ CS5+ CS6+ CS4+ CS6+ CS5+ CS6+ Uncharacterized Uncharacterized Uncharacterized
A B B C C C C D
C D E E F F F
a Origins: A, strain H10407+ (provided by D. G. Evans, Houston, Tex.); B, Dhaka, Bangladesh (14); C, Dhaka, Bangladesh (5); D, Argentina (provided by N. Binzstein, Institute Malbran, Buenos Aires); E, Provided by B. Rowe (Colindale, London, England); F, India (2, 35).
essential for further epidemiological studies of ETEC transmission. We describe here a colony hybridization assay in which we used a cloned fragment of the CFA/I region 1 DNA containing the promoter and part of the cfaA gene (15). The CFA/I gene probe assay was evaluated against an established CFA/I monoclonal antibody (MAb)-based inhibition ELISA.
MATERIALS AND METHODS Bacterial strains. The characteristics of the strains used for calibrating and evaluating the hybridization assay are given in Table 1. The toxin profiles were determined by using an established oligonucleotide colony hybridization assay (36). Strains with previously determined CFA profiles were used for initial calibration of the washing conditions of the hybridization assays. CFA/I was identified previously by a MAbbased ELISA (21); and CS1, CS2, and CS3 (21a) and CS4 and CS5 (unpublished data) were identified by both ELISA and slide agglutination tests. CS6 was identified previously by using polyclonal antisera in a double-diffusion assay (38). In addition, for evaluating the CFA/I probe, 105 previously uncharacterized ETEC isolates obtained from Indian children with acute and persistent diarrhea (2, 35) were studied. Thus, a total of 135 human ETEC isolates were used to test the characteristics of the CFA/I hybridization assay. In addition, a strain harboring pBS (Stratagene, La Jolla, Calif.), the plasmid vector of the CFA/I probe, was examined to exclude the possibility of probe contamination by vector DNA. For further evaluation of the specificity of the probe, we included eight ETEC strains isolated from animals with diarrhea as well as nine non-ETEC isolates from healthy children. Cultivation of bacteria and inoculation of nitrocellulose membranes. All strains were grown overnight on MacConkey agar at 37°C. Three clones from each strain were examined in parallel by the CFA/I inhibition ELISA and by colony hybridization. To avoid discrepancies caused by the spontaneous loss of CFA/I genes (34), the same three clones from each bacterial isolate were inoculated on sepa-
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rate CFA agar plates (8) and onto nitrocellulose membranes
(Schleicher & Schuell, Inc., Keene, N.H.) overlying fresh MacConkey agar, following the guidelines described earlier (36). The CFA agar plates were incubated overnight at 37°C, while the nitrocellulose membranes were processed as described previously (36). Probe preparation and labeling. pIVB3-c17, a pBS derivative containing a DNA fragment between the first PstI and EcoRI sites of CFA/I region 1 DNA (15), was cleaved with PstI and EcoRI, and the resulting fragments were electrophoretically separated on a 1% agarose gel. The larger of the two fragments, approximately 1,000 base pairs, was isolated for use as the CFA/I probe with the Geneclean kit (Bio 101 Inc., La Jolla, Calif.) according to the instructions of the manufacturer. Similarly, to detect the presence of the regulatory gene, the EcoRI-XbaI fragment of CFA/I region 2, which contains the gene encoding the regulatory protein (28), was used as a probe, termed cfaD. Fifty nanograms of each probe was radiolabeled with 32P as described by Feinberg and Vogelstein (12). A specific activity of 1.5 x 109 cpm/,ug was achieved. Colony hybridization and calibration of washing conditions. Colony hybridization was performed as described earlier (35). Initially, the 30 strains with known CFA profiles were used to determine the optimal washing conditions (33). Thus, after radiolabeling and hybridization, the salt concentration was decreased stepwise from 3x to 0.05x SSC (lx SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate) by using a constant temperature of 67°C. CFA/I inhibition ELISA. The capacity of CFA/I-carrying bacteria to inhibit the binding of anti-CFA/I MAbs to solidphase-bound CFA/I was assayed by a previously described ELISA (21). In brief, ELISA microdilution plates (Nunc, A/S Roskilde, Denmark) were coated with 0.5 p.g of purified CFA/I per ml, 100 ,ul per well, at 37°C overnight. After the plates were washed in phosphate-buffered saline (PBS; pH 7.2), they were blocked with 0.1% bovine serum albumin (BSA) in PBS (PBS-BSA) at 37°C for 30 min. Bacterial suspensions were made by dissolving a loopful of bacteria cultured on CFA agar in 100 ,ul of PBS-BSA. Sixty microliters of each bacterial suspension was added to individual, blocked wells. Anti-CFA/I MAb 1:6 (21) diluted 1/100 in PBS-BSA was added (60 pl per well), and the plates were incubated for 90 min at room temperature. After washing in PBS with 0.05% Tween 20 (PBS-Tween), 100 pul of antimouse immunoglobulin-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories Inc.) diluted 1/2,000 in PBS-Tween-BSA was added per well. After incubation for an additional 90 min, the plates were developed with an o-phenylenediamine-H202 enzyme substrate. Interpreting the test results of CFA/I colony hybridization and ELISA. To minimize bias, the tests were read independently by two investigators before the reference code was broken. Bacterial suspensions that caused 250% inhibition in the ELISA were considered positive. The results were read both visually and spectrophotometrically. The ELISA and hybridization results were interpreted by predefined criteria (36). Thus, a strain was considered CFA/I negative if two or three colonies showed adequate growth and the test results for these were negative. If at least one of the colonies gave a positive result, the strain was considered CFA/I positive regardless of growth pattern. If two of the colonies did not show adequate growth and the third colony gave a negative test result, both assays would be repeated. Examination of strains testing positive in the CFA/I hybridization assay. All strains that tested positive in the CFA/I
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GREWAL ET AL.
TABLE 2. Parallel examination of 135 ETEC strains by CFA/I inhibition ELISA and CFA/I gene probe hybridization Phenotype by ELISA
CFA/I+ CFA/I-
FIG. 1. Autoradiograms of two nitrocellulose membranes containing DNA from the same three ETEC strains after hybridization with a 32P-labeled CFA/I gene probe. The vertical sequence of inoculation starts from the top of the far left column. Five control strains of E. coli (H10407+ harboring the CFA/I gene, a strain with the probe vector, a non-ETEC strain, a CS5+ CS6+ strain, and a CS4+ CS6+ strain) were inoculated onto column 1. Three test strains (18 colonies from each) were examined in columns 2 to 4, 5 to 7, and 8 to 10, respectively. After hybridization, stringent washing of both membranes was performed at 67°C but with different ionic concentrations. The autoradiograms in panels A and B were obtained after washing in lx SSC-0.1% SDS and 0.07x SSC-0.1% SDS, respectively. Strains 1 and 3 (columns 2 to 4 and 8 to 10) yielded clones that produced CFA/I, while clones from strain 2 (columns 5 to 7) were previously shown to produce CS4. The CS4 gene was only detected after low-stringency washing (A), whereas the CFA/I gene was also identified after high-stringency washing (B). Probe-negative clones in columns 3, 4, 7, and 9 probably lost their CFA genes spontaneously. These clones did not produce CFA/I or CS4.
hybridization assay were further examined with the cfaD probe. Strains that showed discrepant results between CFA/I hybridization and ELISA were examined further. Thus, such isolates were again cultured from the original stab agars. Revived clones were tested along with the initially examined clones by slide agglutination using antiCFA/I MAb 1:6 (21). From each isolate, two agglutinating and four nonagglutinating clones were examined by both genotypic assays. RESULTS Standardization of washing conditions. After stringent washing of the hybridized CFA/I gene probe in a solution of lx SSC-0.1% sodium dodecyl sulfate (SDS), some of the colony lysates yielded signals which gradually disappeared when the stringency was increased. These signals disappeared completely after the probe was washed in 0.07x SSC-0.1% SDS, while other signals maintained their intensities (Fig. 1). The strains that were positive on low-stringency washing were previously shown to produce either CFA/I or CS4 (Table 1). After washing at high stringency, only CFA/I strains were detected (Fig. 1). Thus, for evaluating the efficiency of the CFA/I probe, the stringent washes were performed in 0.07x SSC-0.1% SDS at 67°C. Similar calibration of the cfaD probe assay indicated an optimum signal/background ratio after the probe was washed with 0.lx SSC-0.1% SDS at 67°C. No hybridization was observed between the CFA/I probe and DNA from the CS1+ CS3+-, CS2+ CS3+-, CS3+-, or CS5+ CS6+-producing strains, even after low-stringency washing. The eight ETEC strains of animal origin, the 9 non-ETEC strains, and the strain harboring the plasmid vector of the polynucleotide gene probe were all negative in the CFA/I hybridization assay.
No. with the following genotype by hybridization:
CFA/I+
CFA/I-
23 1
111
0
Detection of CFA/I-bearing ETEC by CFA/I ELISA and colony hybridization. Analyses of the 135 human ETEC isolates showed a high level of concordance between the CFA/I ELISA (both visually and spectrophotometrically) and the CFA/I hybridization assay (Table 2). Of the 10 previously characterized CFA/I strains, 8 tested positive in the ELISA as well as in the hybridization assay. However, strain 251575-2 was negative in both assays. Furthermore, all three clones of strain 254175-2, while negative in the ELISA, yielded positive signals in the hybridization assay. Similarly, one of three clones of the Indian strain Bh3-10 showed inconsistency in the gene probe assay and the ELISA. Thus, whereas the DNAs from all three clones hybridized with the CFA/I probe, only two of the clones produced CFA/I, as determined by ELISA. This strain was, by our predefined criteria, typed as CFA/I positive in both assays. Of the remaining 104 Indian ETEC strains, 14 were concomitantly positive in the genotypic and phenotypic CFA/I assays. Thus, altogether, 23 strains tested positive in the CFA/I ELISA, whereas 24 tested positive in the CFA/I hybridization assay. Of the 72 (24 x 3) CFA/I probe-positive clones, only the 4 (3 from strain 254175-2 and 1 from strain Bh3-10) that did not produce CFA/I were negative in the cfaD hybridization assay. Additional clones of these two strains were revived and examined by CFA/I slide agglutination. The two agglutinating clones (from strain 254175-2) were positive in both hybridization assays. On the other hand, one of the four nonagglutinating clones tested positive in the CFA/I hybridization assay, whereas it was negative in the cfaD hybridization assay. The remaining three CFA/I- clones were negative in both gene probe assays. Identical results were obtained on examination of strain Bh3-10. Strain 251575-2, which was previously classified as ST+ LT+ CFA/I+ (14), was found to be negative in both hybridization assays and in the CFA/I ELISA. Repeat examination by the ST probe assay showed that this strain lost its ST gene. In contrast, the four clones (three from strain 254175-2 and one from strain Bh3-10) that were positive in the CFA/I hybridization assay but negative in the cfaD hybridization assay and the CFA/I ELISA maintained their ST genes.
DISCUSSION Parallel examination of 135 ETEC strains with the CFA/I probe and in the CFA/I ELISA showed a high level of correspondence in the identification of strains expressing CFA/I. However, all three clones of one strain were positive in the CFA/I hybridization assay but negative in the ELISA. Interestingly, this strain was previously shown to produce CFA/I (14). A similar discrepant result was obtained in one of three clones of another strain, with the remaining two clones being correspondingly positive in the genotypic and phenotypic tests for CFA/I. Further examination of these four clones (from two strains) that lacked CFA/I revealed the loss of a regulatory gene component. In contrast, all CFA/I
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producers, including additional clones revived from the same two strains, had an intact regulatory gene. Hence, the discrepant results between the CFA/I hybridization assay and the ELISA may reflect an artificially induced inactivation of the previously active CFA/I gene because of longterm storage and repeated subcultures. This observation suggests that studies of CFAs, especially with ETEC strains stored for long periods, may benefit from the detection ofthe CFA genes directly rather than the detection of the gene products. The finding of a concomitant loss of the ST and the CFA/I genes in one strain corroborates the results of earlier reports on the close link between these two genes (10, 29, 30, 34). Interestingly, after low-stringency washing, the CFA/I probe also detected ETEC strains that were previously shown to produce CS4 (H. Sommerfelt et al., unpublished data). This indicates a considerable nucleotide sequence homology between the 5' end of the CFA/I region 1 DNA and CS4-associated nucleotide sequences, thereby implying a close genetic relationship between these two fimbrial structures. This interpretation is supported by the amino acid sequence homology observed between the N-terminal ends of CFA/I and CS4 (42) and the strong immunological cross-reactivity between these two fimbrial antigens (23). In epidemiological studies on the transmission of enteric pathogens, the CFA/I probe evaluated in this study and future probes for other host-specific colonization factors offer a distinct advantage in distinguishing among human and animal ETEC strains. Nonradioactive labeling (33) of such probes will further facilitate characterization of ETEC strains. The application of such probes in epidemiological studies should provide the information needed for future construction of vaccines against diarrhea caused by ETEC strains. ACKNOWLEDGMENTS We thank Curt Endressen and Dag Helland, Center of Biotechnology, as well as Claus Ola Solberg, Medical Department B, University of Bergen, for providing excellent laboratory facilities. The skillful technical assistance of Vibecke Asphaug is gratefully appreciated. The financial support of the Norwegian Research Council for Science and the Humanities, the Norwegian Ministry of Development Cooperation, and the Swedish Medical Research Council is gratefully appreciated. LITERATURE CITED 1. Âhrén, C. M., L. Gothefors, B. J. Stoll, M. A. Salek, and A.-M. Svennerholm. 1986. Comparison of methods for detection of colonization factor antigens on enterotoxigenic Escherichia
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