Colonization factor antigen CFA/IV (PCF8775) of human enterotoxigenic Escherichia coli: nucleotide sequence of the CS5 determinant.

Human enterotoxigenic Escherichia coli isolates expressing the colonization factor antigen CFA/IV (previously designated PCF8775) produce plasmid-encoded CS5 fimbriae. The nucleotide sequence of the region encoding the major CS5 fimbrial subunit was determined. The subunit is synthesized as a precursor of 203 amino acids (20.85 kDa) with a mature protein of 181 amino acids corresponding to a size of 18.6 kDa. The CS5 subunit shows homology to the corresponding component of porcine enterotoxigenic E. coli F41, particularly within the signal sequence and at the carboxy terminus.     

Genetic control and properties of coli surface antigens of colonization factor antigen IV (PCF8775) of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli producing coli surface antigen 4 (CS4), CS5, and CS6 of colonization factor antigen IV were examined. This factor was originally called putative colonization factor 8775 (PCF8775). All of the coli surface antigens were plasmid coded and were usually carried on the same plasmid as the genes coding for heat-stable toxin (ST) or heat-labile toxin (LT); thus, CS5-CS6-ST, CS6-ST, and CS6-LT plasmids were found. In strains of serotype O25:H42, the genes coding for CS4 and CS6 were on a plasmid separate from that containing the genes coding for ST and LT. CS4 and CS5 were fimbrial antigens with a subunit molecular mass of about 17.0 and 21.0 kilodaltons (kDa), respectively. CS6 was found as a single polypeptide with a molecular mass of about 14.5 kDa in strains of serotypes O25:H42, O27:H7, and O27:H20 when heated extracts were run on sodium dodecyl sulfate-polyacrylamide gels. CS6-positive extracts of strains of serogroups O148, O159, and O167 showed two bands with molecular masses between 14.5 and 16.0 kDa.     

New fimbrial antigenic type (E8775) that may represent a colonization factor in enterotoxigenic Escherichia coli in humans.

An enterotoxigenic strain of Escherichia coli O25:H42 (strain E8775), isolated from a patient in Bangladesh with diarrhea, caused mannose-resistant hemagglutination (MRHA) of human and bovine erythrocytes. The strain did not show slide agglutination or immunodiffusion precipitin lines with antiserum specific for the colonization factor antigen CFA/I or CFA/II. A variant E. coli strain, E8775-B, did not cause MRHA or produce enterotoxin. Electron microscopy revealed the presence of fimbriae on the surface of strain E8775 but not strain E8775-B. When strain E8775 was grown at 22 degrees C, it became MRHA negative and fimbriae were absent. An antiserum prepared against strain E8775 was absorbed with strain E8775-B to make an antiserum specific for the fimbrial antigen. Using this absorbed antiserum, we found the fimbrial antigen in 48 of 742 enterotoxigenic E. coli strains. The 48 strains belonged to serogroups O25, O115, and O167. It is suggested by analogy to the properties of previously described colonization factors that these fimbriae may play a part in the colonization of the intestinal epithelium.     

In strains of Escherichia coli O167 a single plasmid encodes for the coli surface antigens CS5 and CS6 of putative colonization factor PCF8775, heat-stable enterotoxin, and colicin Ia.

Twenty Escherichia coli strains of serogroup O167 were examined. They all produced the two surface antigens CS5 and CS6 of the putative colonization factor PCF8775, together with heat-stable enterotoxin and colicin Ia. A plasmid coding for CS5, CS6, heat-stable enterotoxin, and colicin Ia was demonstrated in each strain.     

Role of PCF8775 antigen and its coli surface subcomponents for colonization, disease, and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits.

The role of the PCF8775 antigen and its antigenic subcomponents, in particular, the coli surface (CS) antigen CS6, as colonization factors and protective antigens was studied in the reversible intestinal tie adult rabbit diarrhea model. This was done by testing the abilities of different mutants which carried one or two of the CS components to colonize the intestine and to induce protective immunity against reinfection with PCF8775-positive enterotoxin-producing Escherichia coli. Infection with enterotoxigenic E. coli carrying CS4-CS6, CS5-CS6, or CS6 alone induced diarrhea in 75% or more of the rabbits, whereas the corresponding nonenterotoxigenic mutants, as well as enterotoxigenic but CS-negative strains, induced diarrhea in only a few cases. Mutants carrying CS6 alone colonized the intestine equally as well as strains carrying CS4-CS6 or CS5-CS6 did, whereas CS-negative mutants were excreted in the stool for a significantly shorter period. Rabbits previously infected with mutants carrying CS6 alone or CS6 in combination with CS4 or CS5 developed diarrhea with a significantly lower frequency after reinfection with a normally highly diarrheagenic dose of enterotoxigenic CS4-CS6-positive E. coli bacteria than did animals immunized with corresponding CS-negative mutants. Fecal excretion of the rechallenge strain was also of considerably shorter duration than that observed after initial infection with corresponding strains in 27 of the 30 animals (90%) immunized with strains carrying CS6 alone or in combination with CS4 or CS5. Such reduced shedding of the challenge strain was only seen in a few rabbits (3 of 12) initially infected with CS-negative bacteria. These results suggest that the CS6 component of PCF8775 is a colonization factor in rabbits and that it is also capable of inducing protective immunity.     

Plasmid-encoded production of coli surface-associated antigen 1 (CS1) in a strain of Escherichia coli serotype O139.H28

Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype O139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3. The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype O6.H16 of appropriate biotype. A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E. coli strain HB101 or a derivative of strain E24377 without large plasmids. Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced. Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae. Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence. A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype O6.H16 biotype C carrying plasmid pDEP20.     

An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells.

A genetic determinant related to that encoding the F41 fimbrial adhesin was cloned from a bovine septicemic isolate of Escherichia coli. This determinant was found to mediate expression of morphologically distinct fimbriae in E. coli HB101. The gene encoding the fimbrial subunit protein was identified, and the nucleotide sequence was determined. Homology with the amino-terminal amino acid sequence of CS31A (J. Girardeau, M. Der Vartanian, J. Ollier, and M. Contrepois, Infect. Immun. 56:2180-2188, 1988) was observed, suggesting that this determinant encodes expression of the CS31A fimbrial antigen. The CS31A subunit gene was found to share extensive homology in its signal sequence to the subunit genes encoding the F41 and K88 adhesins. No apparent homology between the mature F41 and CS31A subunits was identified. However, substantial relatedness to the K88 fimbrial subunit was observed. Analysis of the protein products encoded by the CS31A, F41, and K88 determinants in maxicells established that despite extensive genetic similarities between the determinants, each encodes a distinct profile of proteins. E. coli HB101 harboring the cloned CS31A determinant was found to adhere to epithelial cells in a tissue culture assay, suggesting a role for CS31A in adherence. A CS31A-specific DNA hybridization probe detected homologous sequences among enterotoxigenic as well as septicemic E. coli isolates from calves.     

Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4.

The ability to colonize the small intestine is essential for the pathogenesis of diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Colonization is mediated by fimbriae (pili), of which there are several antigenically distinct types, including colonization factor antigen I, colonization factor antigen II (CS1, CS2, and CS3), and PCF8775 (CS4, CS5, and CS6). These fimbriae are associated with certain ETEC O serogroups. Serogroup O159 has had no known colonization factor. We found a distinct plasmid-encoded fimbria composed of 19-kilodalton protein subunits associated with ETEC serotype O159:H4. Rabbit antibody against this purified fimbria reacted with a single 19-kilodalton protein band as seen by Western immunoblot of sheared-cell preparations. The rabbit antibody, treated with colloidal-gold-labeled goat anti-rabbit immunoglobulin G, bound specifically to fimbriae when cells were examined with an electron microscope. Of 10 available ETEC O159:H4 strains from Europe, Bangladesh, and Kenya, 6 expressed this type of fimbria; its true prevalence among ETEC strains is unknown. This putative colonization factor of O159:H4 joins other ETEC fimbriae as potentially useful immunogens against human diarrhea.     

Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli.

Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium. We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one). Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns. Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis. At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen. However, CS1-specific antisera did not react with CfaB. No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.     

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.     

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.     

CS31A, a new K88-related fimbrial antigen on bovine enterotoxigenic and septicemic Escherichia coli strains.

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversely, E. coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits.     

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.     

Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12.

A gonococcal gene bank maintained in Escherichia coli K-12 was screened by colony immunoblotting, and a transformant expressing a surface antigen reactive to anti-gonococcal outer membrane antiserum was isolated. The isolate carried a recombinant plasmid, pTME6, consisting of approximately 9 kilobases of Neisseria gonorrhoeae DNA inserted into the BamHI site of pBR322. Surface labeling of E. coli HB101(pTME6) confirmed that the antigen was expressed on the E. coli cell surface. The antigenic material was resistant to proteinase K digestion and sensitive to periodate oxidation, indicating that the material was carbohydrate. Purified lipopolysaccharide (LPS) from HB101(pTME6) produced a unique band on silver-stained polyacrylamide gels that contained immunoreactive material as seen on Western blots of LPS samples. Only two of three E. coli LPS mutant strains carrying pTME6 reacted with the antigonococcal antiserum, suggesting that a certain E. coli core structure is necessary for antigen expression. We conclude that pTME6 contains one or more gonococcal genes encoding an LPS core biosynthetic enzyme(s) which can modify E. coli core LPS to produce a gonococcuslike epitope(s).     

Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans.

Enterotoxigenic Escherichia coli (ETEC) of serotype O6:H16, biotype A, bearing colonization factor antigen II (CFA/II) possesses two distinct coli surface antigens, CS1 and CS3, whereas CFA/II-positive ETEC of serotype O8:H9 manifests only CS3. CS1 has been shown to be fimbrial in nature, but heretofore the morphology of CS3 has not been described. Accordingly, by immune electron microscopy we investigated the morphological characteristics of CS3 on bacterial cells and after purification. CS3 was found to consist of thin (2-nm), flexible, wiry, "fibrillar" fimbriae, visible both on bacteria (O6:H16, biotype A, and O8:H9 strains) and in the pure state. In contrast, CS1 exists as wider (6-nm), rigid fimbriae on the surface of O6:H16, biotype A, strains. By the use of antisera to CS1 and CS3 in immune electron microscopy, immunodiffusion in gel, and immunoblotting techniques, CS1 and CS3 were found to be immunologically as well as morphologically distinct. Six of nine volunteers who developed diarrhea after challenge with an O139:H28 ETEC strain bearing CS1 and CS3 had significant serological rises to purified CS1 and CS3 antigens, suggesting that both antigens are elaborated in vivo, play a role in pathogenesis, and stimulate an immune response.     

The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded.

A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum. A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E. coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E. coli. All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes. Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster. Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid. Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains. A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII. Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E. coli.     

Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli.

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.     

Functional expression of heterologous fimbrial subunits mediated by the F41, K88, and CS31A determinants of Escherichia coli.

F41, K88, and CS31A are fimbrial adhesins associated with enterotoxigenic Escherichia coli. These adhesins are distinct from one another in the composition of their structural subunits and the adherence characteristics associated with their expression. Despite these differences, extensive homology exists between the genetic determinants mediating the expression of these adhesins, extending throughout the region of each determinant encoding the accessory proteins involved in adhesin biogenesis. This suggests that the regulatory and assembly systems mediating expression of these adhesins may be functionally interchangeable. In the present study we demonstrated that the accessory systems of the F41, K88, and CS31A determinants are able to mediate the functional expression of heterologous fimbrial subunit proteins. Plasmid constructs containing the isolated fimbrial subunit gene of the F41 or CS31A determinant were prepared and introduced into E. coli harboring the F41, K88, and CS31A accessory genes contained on compatible plasmid vectors. The ability of each of the three accessory systems to mediate stable expression of the F41 or CS31A fimbrial subunit peptide was demonstrated by Western blot (immunoblot) analysis. Functional expression of the F41 or CS31A subunit on the bacterial cell surface was demonstrated by the ability of these proteins to confer mannose-resistant hemagglutination of human erythrocytes or in vitro adherence to epithelial cells, respectively. The accessory system of an unrelated adhesin determinant, F1845, did not mediate functional expression of F41 adherence. Taken together, these data indicate that the genetic determinants mediating expression of the F41, K88, and CS31A adhesins are members of a closely related family and suggest that a mechanism exists in the family for the more rapid divergence of genes encoding antigenic and adhesive determinants.     

Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV.

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.     

Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli.

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.