Enterotoxigenic Escherichia Coli Toxin Research Articles

MecVax supplemented with CFA MEFA-II induces functional antibodies against 12 adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21) and 2 toxins (STa, LT) of enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) strains that produce various adhesins and one or two enterotoxins are the leading causes of children's diarrhea and travelers' diarrhea. MecVax, a multivalent ETEC vaccine candidate, consists of two proteins, an adhesin multiepitope fusion antigen (MEFA) that stimulates antibodies to the seven most important ETEC adhesins (CFA/I and CS1-CS6) and a toxoid fusion antigen which stimulates antibodies against ETEC enterotoxins (heat-labile toxin and heat-stable toxin). CFA MEFA-II, another polyvalent MEFA protein, has been demonstrated to stimulate antibodies to another five important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21). We hypothesize that MecVax coverage and efficacy can be expanded if MecVax could stimulate antibodies to all 12 adhesins. In this study, we supplemented MecVax with CFA MEFA-II, examined broad immunity to the 12 targeted ETEC adhesins and 2 ETEC toxins (STa, LT) in mice, and assessed mouse antibody functions for inhibiting the adherence of the 12 adhesins and neutralizing the enterotoxicity of 2 toxins, thus assessing the potential application of a broadly protective pan-ETEC vaccine. Mice intramuscularly immunized with MecVax and CFA MEFA-II developed robust antibody responses to the 12 ETEC adhesins and 2 toxins; furthermore, mouse serum antibodies showed functional activities against the adherence from each of the targeted adhesins and the enterotoxicity of either toxin. Data also indicated that CFA MEFA-II was antigenically compatible with MecVax. These results demonstrated that the inclusion of CFA MEFA-II further expands MecVax broad immunogenicity and protection coverage, suggesting the feasibility of developing a vaccine against all important diarrheal ETEC strains.IMPORTANCEThere are no vaccines licensed for Enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Since ETEC strains produce over 25 adhesins and 2 distinctive enterotoxins, heterogeneity is a key obstacle to vaccine development. MecVax, a multivalent ETEC vaccine candidate, induces protective antibodies against the seven most important adhesins (CFA/I and CS1-CS6) associated with two-thirds of ETEC clinical cases. However, ETEC prevalence shifts chronically and geographically, and other adhesins are also associated with clinical cases. MecVax would become a pan-ETEC vaccine if it also protects against the remaining important adhesins. This study demonstrated that MecVax supplemented with adhesin protein CFA MEFA-II induces functional antibodies against 12 important ETEC adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21), enabling the development of a more broadly protective ETEC vaccine and further validating the application of the MEFA vaccinology platform for multivalent vaccine development.

The Antimicrobial Resistance of Enterotoxigenic Escherichia coli from Diarrheal Patients and the Environment in Two Geographically Distinct Rural Areas in Bangladesh over the Years.

Antimicrobial resistance (AMR) is an unprecedented global health challenge, involving the transfer of bacteria and genes between humans and the environment. We simultaneously and longitudinally determined the AMR of enterotoxigenic Escherichia coli (ETEC) strains isolated from diarrheal patients and an aquatic environment over two years from two geographically distinct locations, Coastal Mathbaria and Northern Chhatak in Bangladesh. A total of 60% and 72% of ETEC strains from the patients in Mathbaria and Chhatak, respectively, were multi-drug resistant (MDR) with a high proportion of ETEC resistant to nalidixic acid (80.7%), macrolides (49.1-89.7%), ampicillin (57.9-69%), and trimethoprim/sulfamethoxazole (55.2%). From the surface water, 68.8% and 30% of ETEC were MDR in Mathbaria and Chhatak, respectively, with a high proportion of ETEC strains resistant to macrolides (87.5-100%), ampicillin (50-75%), ceftriaxone (62.5%), and nalidixic acid (40%). Notably, 80-100% of the ETEC strains were susceptible to tetracycline and quinolones (ciprofloxacin and norfloxacin), both in clinical and aquatic ETEC. The AMR varied by the ETEC toxin types. The patterns of excessive or limited consumption of drugs to treat diarrhea over time in Bangladesh were reflected in the ETEC AMR from the patients and the environment. The high prevalence of MDR-ETEC strains in humans and the environment is of concern, which calls for vaccines and other preventative measures against ETEC.

Contribution of the Rapid LAMP-Based Diagnostic Test (RLDT) to the Evaluation of Enterotoxigenic Escherichia coli (ETEC) and Shigella in Childhood Diarrhea in the Peri-Urban Area of Ouagadougou, Burkina Faso.

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a "Rapid LAMP based Diagnostic Test (RLDT)" to detect ETEC and Shigella in diarrheal and non-diarrheal stool samples from a 12-month longitudinal cohort of children under five years of age in a peri-urban area of Ouagadougou in Burkina Faso (West Africa). To allow comparison with the RLDT-Shigella results, conventional culture methods were used to identify Shigella strains in the stool samples. As conventional culture alone cannot detect ETEC cases, a subset of E. coli-like colonies was tested using conventional PCR to detect ETEC toxins genes. Of the 165 stool samples analyzed for ETEC, 24.9% were positive when using RLDT against 4.2% when using culture followed by PCR. ETEC toxin distribution when using RLDT was STp 17.6% (29/165), LT 11.5% (19/165), and STh 8.5% (14/165). Of the 263 specimens tested for Shigella, 44.8% were positive when using RLDT against 23.2% when using culture. The sensitivity and specificity of the RLDT compared to culture (followed by PCR for ETEC) were 93.44% and 69.8% for Shigella and 83.7% and 77.9% for ETEC, respectively. This study indicates that both Shigella and ETEC are substantially underdiagnosed when using conventional culture and highlights the potential contribution of the new RLDT method to improve enteric disease burden estimation and to guide future efforts to prevent and control bacterial enteric infection and disease.

Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh.

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

A Polyvalent Adhesin-Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa).

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children's diarrhea and travelers' diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins.

Intramuscularly Administered Enterotoxigenic Escherichia coli (ETEC) Vaccine Candidate MecVax Prevented H10407 Intestinal Colonization in an Adult Rabbit Colonization Model.

ABSTRACTCurrently, there are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of children’s diarrhea in developing countries and the most common cause of travelers’ diarrhea. A vaccine preventing ETEC bacteria from colonization at small intestines and neutralizing enterotoxin toxicity is expected to be effective against ETEC diarrhea. Protein-based multivalent vaccine candidate MecVax was demonstrated recently to induce antibodies neutralizing heat-labile toxin (LT) and heat-stable toxin (STa) enterotoxicity and inhibiting adherence of seven ETEC adhesins (CFA/I, CS1 to CS6) but also to protect against ETEC toxin-mediated clinical diarrhea in a pig challenge model. To further evaluate MecVax preclinical efficacy against ETEC colonization at small intestines, in this study, we intramuscularly immunized adult rabbits with MecVax, challenged rabbits with ETEC strain H10407 (CFA/I, LT, STa), and examined prevention of bacteria intestinal colonization. Data showed that rabbits immunized with MecVax developed antibodies to both ETEC toxins (LT, STa) and seven adhesins (CFA/I, CS1 to CS6) and had over 99.9% reduction of H10407 intestinal colonization, indicating that the broadly immunogenic ETEC vaccine candidate MecVax is protective against ETEC H10407 intestinal colonization. This study also confirmed that parenteral administration of a protein-based vaccine can prevent bacteria intestinal colonization. Protection against ETEC intestinal colonization demonstrated by this rabbit study, in conjugation with protection against ETEC enterotoxin-mediated clinical diarrhea from a previous pig challenge study, suggested that MecVax can potentially be an effective ETEC vaccine and a combined pig and rabbit challenge model can evaluate ETEC vaccine preclinical efficacy.IMPORTANCE An effective ETEC vaccine would prevent hundreds of millions of diarrhea clinical cases and save nearly 100,000 lives annually. MecVax, a protein-based injectable multivalent ETEC vaccine candidate, has been shown for the first time to induce functional antibodies against both ETEC enterotoxins (STa, LT) produced by all ETEC strains and seven ETEC adhesins (CFA/I, CS1 to CS6) expressed by ETEC strains causing a majority of ETEC diarrhea clinical cases and the moderate-to-severe cases. Moreover, MecVax was demonstrated to protect against ETEC STa or LT toxin-mediated diarrhea in a pig model. If it also protects against ETEC intestinal colonization, MecVax can be validated as an effective ETEC vaccine candidate. This adult rabbit colonization model study showed that intramuscular administration of MecVax effectively prevented intestinal colonization by H10407, perhaps the most virulent ETEC strain, affirming MecVax vaccine candidacy and accelerating vaccine development against ETEC children’s diarrhea and travelers’ diarrhea.

Evaluation of the Safety, Tolerability and Immunogenicity of ShigETEC, an Oral Live Attenuated Shigella-ETEC Vaccine in Placebo-Controlled Randomized Phase 1 Trial.

Background: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) cause high morbidity and mortality worldwide, yet no licensed vaccines are available to prevent corresponding infections. A live attenuated non-invasive Shigella vaccine strain lacking LPS O-antigen and expressing the ETEC toxoids, named ShigETEC was characterized previously in non-clinical studies. Methods: ShigETEC was evaluated in a two-staged, randomized, double-blind and placebo-controlled Phase I clinical trial. A single dose of increasing amounts of the vaccine was given to determine the maximum tolerated dose and increasing number of immunizations were administered with an interval based on the duration of shedding observed. Results: Oral immunization with ShigETEC was well tolerated and safe up to 4-time dosing with 5 × 1010 colony forming units. ShigETEC induced robust systemic immune responses against the Shigella vaccine strain, with IgA serum antibody dominance, as well as mucosal antibody responses evidenced by specific IgA in stool samples and in ALS (Antibodies in Lymphocyte Supernatant). Anti- ETEC toxin responses were detected primarily in the 4-times immunized cohort and for the heat-labile toxin correlated with neutralizing capacity. Conclusion: ShigETEC is a promising vaccine candidate that is scheduled for further testing in controlled human challenge studies for efficacy as well as in children in endemic setting for safety and immunogenicity.

54 Evaluating the effectiveness of Lactobacillus zeae against enterotoxigenic Escherichia coli F4 infection in a porcine intestinal epithelial cell model

Abstract Lactobacillus zeae (LB1) has been shown to prevent Caenorhabditis elegans from death caused by enterotoxigenic Escherichia coli (ETEC) infection through downregulating the gene expression of ETEC toxins and mediating antimicrobial peptides/defense molecules under the p38 MAPK and DAF/IGF signaling pathways in the nematode. However, the protective effects of LB1 at a cellular level have not been investigated yet. In the present study, porcine intestinal cells (IPEC-J2) were used to examine the potential of LB1 on modulating intestinal barrier and innate immune functions and protecting against intestinal injuries and inflammatory reactions induced by ETEC F4 infection. The results showed that the pretreatment of IPEC-J2 cells with LB1 significantly alleviated the cytotoxicity and inhibited gene expression of interleukin (IL)-8 and IL-6, and secretion of IL-8 induced by ETEC F4 (P < 0.05). The LB1 pretreatment significantly maintained a higher value of TEER concomitantly with lower dextran-fluorescein fluxes from the apical side to the basolateral side when compared with that challenged by ETEC F4. LB1 pretreatment also prevented morphological damage of tight junction and cytoskeleton caused by the ETEC F4 challenge, indicating that LB1 pretreatment maintained the structural integrity of tight junction. However, LB1 inclusion showed no significant protection on ZO-1 expression at both gene and protein levels from the ETEC F4 challenge, but a considerable increase in the protein expression of occludin (P < 0.05). LB1 administration influenced neither ETEC F4 adhesion nor secretion of antimicrobial defensin (pBD2, pBD3), while dramatically reduced the mRNA level of toll-like receptor 4 (TLR4). These results suggested that probiotics LB1 could effectively protect the mucosa from ETEC F4 infection by inhibiting inflammation and maintaining barrier integrity via downregulating TLR4 expression. Our data provide further evidence on the mechanisms at the cellular level that probiotics could improve pig gut health.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Anti-infectious properties of the probiotic Saccharomyces cerevisiae CNCM I-3856 on enterotoxigenic E. coli (ETEC) strain H10407.

Enterotoxigenic Escherichia coli (ETEC) are major food-borne pathogens responsible for traveler's diarrhea. The production of adhesins and the secretion of enterotoxins constitute the major virulence traits of the bacteria. Treatments are mainly symptomatic and can involve antibiotherapy. However, given the rise of antibiotic resistance worldwide, there is an urgent need for the development of new preventive strategies for the control of ETEC infections. Among them, a promising approach is the use of probiotics. The aim of this study was to investigate, using complementary in vitro and in vivo approaches, the inhibitory potential of the yeast Saccharomyces cerevisiae CNCM I-3856 against the human ETEC reference strain H10407. In conventional culture media, S. cerevisiae significantly reduced ETEC growth and toxin production. The yeast also inhibited bacterial adhesion to mucin-agar and intestinal Caco-2/TC7 cells in a dose-dependent manner. Lastly, pre-treatment with S. cerevisiae inhibited interleukin-8 production by ETEC-infected intestinal cells. In streptomycin-treated mice, the probiotic yeast decreased bacterial colonization, mainly in the ileum, the main site of ETEC pathogenesis. For the first time, this study shows that the probiotic yeast S. cerevisiae CNCM I-3856 can exert an anti-infectious activity against a human ETEC strain through a multi-targeted approach, including inhibition of bacterial growth and toxin production, reduction of bacterial adhesion to mucins and intestinal epithelial cells, and suppression of ETEC-induced inflammation. Interestingly, the highest activity was obtained with a prophylactic treatment. Further studies will aim to assess the effect of the yeast on ETEC survival and virulence under human simulated digestive conditions.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, ‘LTR192G-STb-Stx2e-STaP12F’ and ‘LTR192G-STb-Stx2e-3xSTaP12F’ which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with ‘LTR192G-STb-Stx2e-3xSTaP12F’ were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

Generation and Characterization of a Live Attenuated Enterotoxigenic Escherichia coli Combination Vaccine Expressing Six Colonization Factors and Heat-Labile Toxin Subunit B

Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, double-blind placebo-controlled phase II challenge trial, both in healthy adult volunteers.

Survival and gene expression of enterotoxigenicEscherichia coliduring long-term incubation in sea water and freshwater

In this study, the main objective was to verify the hypothesis of induction of 'viable but non-culturable' (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat-labile (LT) or heat-stable (ST) enterotoxins was observed using GM1-ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real-time RT-PCR on cDNA derived from the bacteria. Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long-term incubation in water.

Development of Multiplex PCR Assays for Detection of Enterotoxigenic Escherichia coli Colonization Factors and Toxins

Four multiplex PCR assays for detection of 19 enterotoxigenic Escherichia coli (ETEC) colonization factors and an improved ETEC toxin multiplex PCR were developed and tested on Bangladeshi and Bolivian ETEC strain collections. The assays will be useful for surveillance of ETEC infections in diagnostic laboratories that have access to PCR.

Vaccines against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children less than 3 years of age in developing countries and in travelers to these areas. The key pathogenic mechanisms that contribute to the pathogenesis of ETEC are the production of colonization factors (CFs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin. To provide broad-spectrum protection, an ETEC vaccine should, most likely, contain the most prevalent fimbrial antigens, that is, CF antigen I and CS1–CS6, and/or a LT toxoid. Different strategies have been taken to deliver ETEC fimbriae and toxin antigens to the human immune system to elicit strong mucosal, in particular, intestinal immune responses that are considered to be of prime importance for protection against ETEC disease. There has been some promise when testing different ETEC candidate vaccines for protection against diarrhea in adult travelers. However, no ETEC candidate vaccine has been shown to be effective in the most important target group, which is infants and young children in endemic areas. Against this background, intense efforts are in progress to try to improve the immunogenicity of different available candidate vaccines, as well as to develop new types of ETEC vaccines.

Epidemiology of enterotoxigenic Escherichia coli diarrhea in a pediatric cohort in a periurban area of lower Egypt.

Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype. In total, 242 Egyptian children aged <3 years were prospectively followed in 1993-1995. ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identified from monthly cross-sectional surveys. ETEC episodes were 0.6 per child-year. ST-only ETEC was 2.6 times (P<.001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation. Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes. Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea. These findings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation.

An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli.

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.