Pathogenicity and immune response measured in mice following intranasal challenge with enterotoxigenic Escherichia coli strains H10407 and B7A

The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.     

Refinement of a human challenge model for evaluation of enterotoxigenic Escherichia coli vaccines

Enterotoxigenic Escherichia coli (ETEC) strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 (serotype O78:H11 producing heat-labile toxin [LT], heat-stable toxin [ST], and colonization factor I [CFA/I]) induces reliably high diarrheal attack rates (ARs) in a human challenge model at doses of ≥10⁹ CFU. A descending-dose challenge study was conducted with changes to the standard fasting time and buffer formulation, seeking conditions that permit lower inocula while maintaining reproducibly high ARs. In cohort 1, 20 subjects were fasted overnight and randomized 1:1:1:1 to receive {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 at doses of 10⁸ CFU with bicarbonate, 10⁸ CFU with CeraVacx, 10⁷ CFU with bicarbonate, or 10⁷ CFU with CeraVacx. Subsequent cohorts received {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 (10⁷ CFU with bicarbonate) with similar fasting conditions. Cohort 2 included 15 ETEC-naïve volunteers. Cohort 3 included 10 ETEC-naïve volunteers and 10 rechallenged volunteers. In all, 25/35 (71%) ETEC-naïve recipients of 10⁷ CFU of {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 developed moderate or severe diarrhea (average maximum stool output/24 h = 1,042 g), and most (97%) shed {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 (maximum geometric mean titer = 7.5 × 10⁷ CFU/gram of stool). Only one of 10 rechallenged volunteers developed diarrhea. These rechallenged subjects had reduced intestinal colonization, reflected by quantitative microbiology of fecal samples. Among the 35 ETEC-naïve subjects, anti-lipopolysaccharide (LPS) O78 serum antibody responses were striking, with positive IgA and IgG antibody responses in 33/35 (94%) and 25/35 (71%), respectively. Anti-heat-labile enterotoxin (LTB) serum IgA and IgG responses developed in 19/35 (54%) and 14/35 (40%) subjects, respectively. Anti-CFA/I serum IgA and IgG responses were detected in 15/35 (43%) and 8/35 (23%) subjects. After the second challenge, participants exhibited blunted anti-LPS and -LTB responses but a booster response to CFA/I. This ETEC model should prove useful in the future evaluation of ETEC vaccine candidates.     

The Oral,Live Attenuated Enterotoxigenic Escherichia coli Vaccine ACE527 Reduces the Incidence and Severity of Diarrhea in a Human Challenge Model of Diarrheal Disease

An oral, live attenuated, three-strain recombinant bacterial vaccine, ACE527, was demonstrated to generate strong immune responses to colonization factor and toxin antigens of enterotoxigenic Escherichia coli (ETEC) in human volunteers. The vaccine was safe and well tolerated at doses of up to 10¹¹ CFU, administered in each of two doses given 21 days apart. These observations have now been extended in a phase 2b study with a total of 70 subjects. Fifty-six of these subjects were challenged 28 days after the second dose of vaccine with the highly virulent ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 to obtain preliminary indicators of efficacy against disease and to support further development of the vaccine for both travelers and infants in countries where ETEC is endemic. The vaccine had a significant impact on intestinal colonization by the challenge strain, as measured by quantitative fecal culture 2 days after challenge, demonstrating the induction of a functional immune response to the CFA/I antigen. The incidence and severity of diarrhea were also reduced in vaccinees as measured by a number of secondary and ad hoc endpoints, although the 27% reduction seen in the primary endpoint, moderate to severe diarrhea, was not statistically significant. Together, these observations support the hypothesis that the ACE527 vaccine has a dual mode of action, targeting both colonization factors and the heat-labile enterotoxin (LT), and suggest that it should be further developed for more advanced trials to evaluate its impact on the burden of ETEC disease in field settings.     

Antibody-secreting cells in human peripheral blood after oral immunization with an inactivated enterotoxigenic Escherichia coli vaccine.

Vaccine antigen-specific antibody-secreting cell (ASC) responses in peripheral blood of healthy adult volunteers were studied after oral immunization with a prototype enterotoxigenic Escherichia coli (ETEC) vaccine by means of the enzyme-linked immunospot technique. Three doses of vaccine consisting of formalin-killed ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) in combination with purified cholera toxin B subunit (CTB) were given 2 weeks apart. The ASC responses were detected 7 days after each immunization. Immunoglobulin A (IgA) was the predominant isotype produced by CFA/I- as well as CFA/II-specific ASCs. Moderate CFA/I- and CFA/II-specific IgM-secreting ASC (IgM-ASC) responses were also seen, whereas IgG-ASC responses to either of the CFAs were negligible. The ASC responses to CTB, on the other hand, comprised both IgA- and IgG-ASCs, with few if any specific IgM-ASCs. Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination. Maximal CFA-specific ASC responses were usually observed after a single dose or two doses of vaccine. A third dose of vaccine did not result in increased but rather resulted in decreased magnitudes of CFA-specific ASC responses. Furthermore, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB. These results suggest that two oral doses of ETEC vaccine induce a strong mucosal immune response, as reflected by the presence of large numbers of antigen-specific mucosal B cell immunoblasts in the blood.     

Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic escherichia coli-cholera toxin B subunit vaccine in Israeli young adults

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units. Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB). The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers. However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms. Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively. The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1. The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens. The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens. Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force.     

Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers'' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10⁷ bacteria resulted in 50% lethal doses (LD₅₀) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD₅₀ challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.     

A combination vaccine consisting of three live attenuated enterotoxigenic Escherichia coli strains expressing a range of colonization factors and heat-labile toxin subunit B is well tolerated and immunogenic in a placebo-controlled double-blind phase I trial in healthy adults

Immune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whose aroC, ompC, and ompF genes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 10(10) and 10(11) total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.     

Dose-dependent circulating immunoglobulin A antibody-secreting cell and serum antibody responses in Swedish volunteers to an oral inactivated enterotoxigenic Escherichia coli vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli can prime and boost immune responses against heterologous CFAs

The fimbrial colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli (ETEC) are antigenically different, but have closely related N-terminal amino acid sequences. We have studied the capacity of purified CFA/I and CS4 fimbriae respectively to prime and boost immune responses against the homologous and heterologous CFAs in parenterally immunized mice. Based on initial characterization of the kinetics of the primary and secondary CFA/I immune responses in serum as well as antibody-secreting cell (ASC) responses in spleen, two doses with different combinations of the purified CFAs were given 7 weeks apart. It was shown, using either assay, that CFA/I could both prime and boost immune responses against CS4, and, conversely, that CS4 could prime and boost immune responses against CFA/I. These findings suggest the presence of immunorecessive epitopes, or epitopes that are not surface-exposed on whole fimbriae, that are shared between CFA/I and CS4 and which can expand B-cell clones with specificity for heterologous fimbriae.     

Homologous and cross-reactive immune responses to enterotoxigenic Escherichia coli colonization factors in Bangladeshi children

We have studied homologous (HoM) and cross-reacting (CR) immunoglobulin A (IgA) antibody responses to colonization factors (CFs) in Bangladeshi children with diarrhea due to enterotoxigenic E. coli (ETEC) strains of the CF antigen I (CFA/I) group (CFA/I, n = 25; coli surface antigen 4 [CS4], n = 8; CS14, n = 11) and the CS5 group (CS5, n = 15; CS7, n = 8), respectively. The responses to the HoM, CR, and heterologous (HeT) CF antigens in each group of patient were studied and compared to that seen in healthy children (n = 20). In the CFA/I group (CFA/I and CS14), patients responded with antibody-secreting cell (ASC) responses to HoM CFs (geometric mean, 156 to 329 ASCs/10(6) peripheral blood mononuclear cells [PBMCs]) and to CR CFs ( approximately 15 to 38 ASCs/10(6) PBMCs) but least of all to the HeT CS5 antigen (2 to 4 ASCs/10(6) PBMCs). For the CS5 group of patients with ETEC (CS5 and CS7), likewise, responses to HoM CFs (230 to 372 ASCs/10(6) PBMCs) and CR CFs (27 to 676 ASCs/10(6) PBMCs) were seen, along with lower responses to the HeT CFA/I antigen (9 to 38 ASCs/10(6) PBMCs). Both groups of patients responded with CF-specific IgA antibodies to HoM and CR antigens in plasma but responded less to the HeT CFs. The responses in patients were seen very soon after the onset of diarrhea and peaked around 1 week after onset. Vaccinees who had received two doses of the oral, killed whole-cell ETEC vaccine (CF-BS-ETEC) responded with plasma IgA antibodies to CFA/I, a component of the vaccine, but also to the CR CS14 antigen, which was not included in the vaccine, showing that antibody responses can be stimulated by a CFA/I-containing ETEC vaccine to a CR-reacting antigen in individuals in countries where ETEC is endemic.     

Intestinal immune responses in patients infected with enterotoxigenic Escherichia coli and in vaccinees

Immune responses against enterotoxigenic Escherichia coli (ETEC) were examined in Bangladeshi adults with naturally acquired disease and compared to responses in age-matched Bangladeshi volunteers who had been orally immunized with a vaccine consisting of inactivated ETEC bacteria expressing different colonization factor antigens (CFs) and the B subunit of cholera toxin. B-cell responses in duodenal biopsy samples, feces, intestinal washings, and blood were determined. Because most of the patients included in the study were infected with ETEC expressing CS5, immune responses to this CF were studied most extensively. Vaccinees and patients had comparable B-cell responses against this antigen in the duodenum: the median numbers of antibody-secreting cells (ASC) were 3,300 immunoglobulin A (IgA) ASC/10(7) mononuclear cells (MNC) in the patient group (n = 8) and 1,200 IgA ASC/10(7) MNC in the vaccinees (n = 13) (not a significant difference). Similarly, no statistically significant differences were seen in the levels of duodenal B cells directed against enterotoxin among vaccinees and patients. A comparison of the capacities of the various methods used to assess mucosal immune responses revealed a correlation between numbers of circulating B cells and antibody levels in saponin extracts of duodenal biopsy samples (r = 0.58; n = 13; P = 0.04) after vaccination. However, no correlation was seen between blood IgA ASC and duodenal IgA ASC after two doses of vaccine. Still, a correlation between numbers of CF-specific B cells in blood sampled from patients early during infection and numbers of duodenal B cells collected 1 week later was apparent (r = 0.70; n = 10; P = 0.03).     

Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407

Enterotoxigenic Escherichia coli (ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 challenge strain. Subjects challenged with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.     

Dose-Dependent Circulating Immunoglobulin A Antibody-Secreting Cell and Serum Antibody Responses in Swedish Volunteers to an Oral Inactivated Enterotoxigenic Escherichia coli Vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Efficacy of enteric-coated protease in preventing attachment of enterotoxigenic Escherichia coli and diarrheal disease in the RITARD model

In this study, we report on a novel approach based on modification of the intestinal surface to prevent diarrhea caused by enterotoxigenic Escherichia coli (ETEC). The removable intestinal tie adult rabbit diarrhea (RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor Technology Pty. Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization factor antigen I-positive (CFA/I+) E. coli H10407. Protease was administered orally to rabbits 18 h prior to challenge with 10(11) bacteria. Four groups of rabbits were inoculated with different ETEC strains which produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline. Occurrence of diarrhea during the subsequent 24-h incubation period was recorded. Oral administration of protease was successful in reducing diarrhea and diarrhea-induced death in six of seven (86%) rabbits infected with CFA/I+, heat-stable and heat-labile toxin-positive E. coli (H10407). Seven of eight (87%) rabbits not protected by protease treatment died or developed severe diarrhea. Quantitative analysis of bacterial cultures obtained from the small intestine of rabbits showed a significant (P less than 0.001) 2,000-fold reduction in CFU per centimeter of intestine following treatment with protease. The efficacy of protease treatment was 99.5%, with very wide confidence limits (greater than 0 to 99.9%). The data indicate that the use of protease to prevent ETEC diarrheal disease has considerable potential.     

Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.     

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.     

Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina.

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 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 O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128: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 O6: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-resistant 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.     

Prevalence of toxin types and colonization factors in enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Bangladesh

The prevalence of toxin types and colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) was prospectively studied with fresh samples (n = 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (n = 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (P < 0.001) or LT- and ST-positive (P < 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.     

Use of nonradioactive DNA hybridization for identification of enterotoxigenic Escherichia coli harboring genes for colonization factor antigen I, coli surface antigen 4, or putative colonization factor O166.

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.     

Plasmids coding for colonization factor antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Escherichia coli.

Colonization factor antigens I and II (CFA/I and CFA/II) are important in the pathogenesis of diarrhea in humans caused by some enterotoxigenic Escherichia coli (ETEC). Plasmid DNA from 16 CFA/I+ and five CFA/II+ ETEC were examined by Southern blot analysis with enterotoxin gene probes and were compared with plasmid DNA from derivatives of the same ETEC that had lost the ability to produce these colonization factors. Among the 16 CFA/I+ ETEC strains, the loss of CFA/I was accompanied by the loss of a plasmid of between 34 and 68 megadaltons (MDa) coding for heat-stable enterotoxin A2 (ST-A2) in 12 strains, by the loss of a 60-MDa plasmid coding for heat-labile enterotoxin (LT) and ST-A2 in one strain, or by deletions of a segment of DNA encoding for ST-A2 in three strains. Among five CFA/II+ ETEC strains, the loss of CFA/II was associated with the loss of a plasmid of 75 MDa coding for LT and ST-A2 in three strains, with the loss of genes coding for LT and ST-A2 from a 68-MDa plasmid in one strain, or with no discernible loss of a plasmid or DNA sequences coding for enterotoxins in the remaining strain. The loss of CFA/I and CFA/II production was associated with the loss of DNA sequences encoding for ST-A2 in 20 of 21 ETEC examined.